Bicyclic peptide ligands specific for Nectin-4

ABSTRACT

The present invention relates to polypeptides which are covalently bound to molecular scaffolds such that two or more peptide loops are subtended between attachment points to the scaffold. In particular, the invention describes peptides which are high affinity binders of Nectin-4. The invention also includes drug conjugates comprising said peptides, conjugated to one or more effector and/or functional groups, to pharmaceutical compositions comprising said peptide ligands and drug conjugates and to the use of said peptide ligands and drug conjugates in preventing, suppressing or treating a disease or disorder mediated by Nectin-4.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 5, 2019, isnamed BIC-C-P2584US_sequence_listing_revised_ST25.txt and is 2,000 bytesin size.

FIELD OF THE INVENTION

The present invention relates to polypeptides which are covalently boundto molecular scaffolds such that two or more peptide loops are subtendedbetween attachment points to the scaffold. In particular, the inventiondescribes peptides which are high affinity binders of Nectin-4. Theinvention also includes drug conjugates comprising said peptides,conjugated to one or more effector and/or functional groups, topharmaceutical compositions comprising said peptide ligands and drugconjugates and to the use of said peptide ligands and drug conjugates inpreventing, suppressing or treating a disease or disorder mediated byNectin-4.

BACKGROUND OF THE INVENTION

Cyclic peptides are able to bind with high affinity and targetspecificity to protein targets and hence are an attractive moleculeclass for the development of therapeutics. In fact, several cyclicpeptides are already successfully used in the clinic, as for example theantibacterial peptide vancomycin, the immunosuppressant drugcyclosporine or the anti-cancer drug octreotide (Driggers et al. (2008),Nat Rev Drug Discov 7 (7), 608-24). Good binding properties result froma relatively large interaction surface formed between the peptide andthe target as well as the reduced conformational flexibility of thecyclic structures. Typically, macrocycles bind to surfaces of severalhundred square angstrom, as for example the cyclic peptide CXCR4antagonist CVX15 (400 Å²; Wu et al. (2007), Science 330, 1066-71), acyclic peptide with the Arg-Gly-Asp motif binding to integrin αVb3 (355Å²) (Xiong et al. (2002), Science 296 (5565), 151-5) or the cyclicpeptide inhibitor upain-1 binding to urokinase-type plasminogenactivator (603 Å²; Zhao et al. (2007), J Struct Biol 160 (1), 1-10).

Due to their cyclic configuration, peptide macrocycles are less flexiblethan linear peptides, leading to a smaller loss of entropy upon bindingto targets and resulting in a higher binding affinity. The reducedflexibility also leads to locking target-specific conformations,increasing binding specificity compared to linear peptides. This effecthas been exemplified by a potent and selective inhibitor of matrixmetalloproteinase 8 (MMP-8), which lost its selectivity over other MMPswhen its ring was opened (Cherney et al. (1998), J Med Chem 41 (11),1749-51). The favorable binding properties achieved throughmacrocyclization are even more pronounced in multicyclic peptides havingmore than one peptide ring as for example in vancomycin, nisin andactinomycin.

Different research teams have previously tethered polypeptides withcysteine residues to a synthetic molecular structure (Kemp and McNamara(1985), J. Org. Chem; Timmerman et al. (2005), ChemBioChem). Meloen andco-workers had used tris(bromomethyl)benzene and related molecules forrapid and quantitative cyclisation of multiple peptide loops ontosynthetic scaffolds for structural mimicry of protein surfaces(Timmerman et al. (2005), ChemBioChem). Methods for the generation ofcandidate drug compounds wherein said compounds are generated by linkingcysteine containing polypeptides to a molecular scaffold as for exampleTATA (1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one, Heiniset al. Angew Chem, Int Ed. 2014; 53:1602-1606).

Phage display-based combinatorial approaches have been developed togenerate and screen large libraries of bicyclic peptides to targets ofinterest (Heinis et al. (2009), Nat Chem Biol 5 (7), 502-7 and WO2009/098450). Briefly, combinatorial libraries of linear peptidescontaining three cysteine residues and two regions of six random aminoacids (Cys-(Xaa)₆-Cys-(Xaa)₆-Cys) were displayed on phage and cyclisedby covalently linking the cysteine side chains to a small moleculescaffold.

SUMMARY OF THE INVENTION

According to a first aspect of the invention, there is provided apeptide ligand specific for Nectin-4 comprising a polypeptide comprisingat least three cysteine residues, separated by at least two loopsequences, and a molecular scaffold which forms covalent bonds with thecysteine residues of the polypeptide such that at least two polypeptideloops are formed on the molecular scaffold, wherein the peptide ligandcomprises the amino acid sequence:

(SEQ ID NO: 1) C_(i)P[1Nal][dD]C_(ii)M[HArg]DWSTP[HyP]WC_(iii);wherein 1Nal represents 1-naphthylalanine, HArg represents homoarginine,HyP represents hydroxyproline and C_(i), C_(ii) and C_(iii) representfirst, second and third cysteine residues, respectively or apharmaceutically acceptable salt thereof.

According to a further aspect of the invention, there is provided a drugconjugate comprising a peptide ligand as defined herein conjugated toone or more effector and/or functional groups.

According to a further aspect of the invention, there is provided apharmaceutical composition comprising a peptide ligand or a drugconjugate as defined herein in combination with one or morepharmaceutically acceptable excipients.

According to a further aspect of the invention, there is provided apeptide ligand or drug conjugate as defined herein for use inpreventing, suppressing or treating a disease or disorder mediated byNectin-4.

BRIEF DESCRIPTION OF THE FIGURES

Where present in the figures, error bars represent standard error of themean (SEM).

FIGS. 1 and 2: Tumor volume traces after administering BCY8245 to femaleBALB/c nude mice bearing NCI-H292 xenograft.

FIGS. 3 and 4: Tumor volume traces after administering BCY8245 to femaleCB17-SCID mice bearing HT-1376 xenograft.

FIG. 5: Tumor volume trace after administering BCY8245 to female Balb/cnude mice bearing Panc2.13 xenograft.

FIG. 6: Tumor volume trace after administering BCY8245 to female Balb/cnude mice bearing MDA-MB-468 xenograft.

FIG. 7: Tumor volume trace after administering BCY8549 (with BCY8245 ascontrol), to female BALB/c nude mice bearing NCI-H292 xenograft.

FIG. 8: Gating strategy for Nectin-4 in Breast (T-47D and MDA-MB-468).

FIG. 9: Gating strategy for Nectin-4 in NCI-H292 and NCI-H322.

FIGS. 10 and 11: Gating strategy for Nectin-4 in NCI-H526 and HT1080,respectively.

FIGS. 12-16: Gating strategy for Nectin-4 in Bladder cancer (HT1376;FIG. 12), Breast cancer (MDA-MB-468; FIG. 13), Colorectal cancer (HT-29;FIG. 14A and HCT-116; FIG. 14B), Lung cancer (A549; FIG. 15A, NCI-H292;FIG. 15B, NCI-H358; FIG. 15C and NCI-526; FIG. 15D), and Pancreas cancer(Panc02.13; FIG. 16), respectively.

FIG. 17: Tumor volume trace after administering BCY8245 to female Balb/cnude mice bearing A549 xenograft.

FIG. 18: Tumor volume trace after administering BCY8245 to female Balb/cnude mice bearing HCT116 xenograft.

FIG. 19: Tumor volume trace after administering BCY8245 to femaleCB17-SCID mice bearing HT-1376 xenograft.

FIG. 20: Tumor volume trace after administering BCY8245 to female Balb/cnude mice bearing MDA-MB-468 xenograft.

FIG. 21: Tumor volume trace after administering BCY8245 to female Balb/cnude mice bearing NCI-H292 xenograft.

FIG. 22: Tumor volume trace after administering BCY8245 to female Balb/cnude mice bearing NCI-H526 xenograft.

FIG. 23: Tumor volume trace after administering BCY8245 to female Balb/cnude mice bearing Panc2.13 xenograft.

FIG. 24: Tumor volume traces after administering BCY8245 or BCY8245 incombination with BCY8234 to female Balb/c nude mice bearing MDA-MB-468xenograft.

FIG. 25: Tumor volume traces after administering BCY8245 alone orBCY8245 in combination with BCY8234 to female Balb/c nude mice bearingMDA-MB-468 xenograft.

FIGS. 26-31: Tumor volume traces in Lu-01-0412, LU-01-0007, CTG-1771,CTG-1171, CTG-1106, and CTG-0896 PDX xenografts.

FIG. 32: BT8009 (i.e. BCY8245) efficacy correlates with expressionCDX/PDX xenografts. Xenografts with little/no Nectin-4 expression showreduced tumour growth rate. Xenografts expressing Nectin-4 showregressions of tumour. Both PDX and CDX models are included in thisanalysis, values are collated from various reports.

FIG. 33: MDA-MB-468 cells express Nectin-4 and show prolonged retentionof MMAE in tumour.

FIG. 34: HCS—Data analysis on MDA-MB-468 cell line.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, the peptide ligand ofC_(i)P[1Nal][dD]C_(ii)M[HArg]DWSTP[HyP]WC_(iii) (SEQ ID NO: 1) comprisesan amino acid sequence selected from:

[B-Ala][Sar10]-(SEQ ID NO: 1)(hereinafter referred to as BCY8234);Ac-[B-Ala][Sar₅]-(SEQ ID NO: 1) (hereinafter referred to as BCY8122);Ac-(SEQ ID NO: 1) (hereinafter referred to as BCY8126);(SEQ ID NO: 1) (hereinafter referred to as BCY8116);Fluorescein-(SEQ ID NO: 1) (hereinafter referred to as BCY8205); and[PYA][B-Ala][Sar10]-(SEQ ID NO: 1) (hereinafter referred to as BCY8846).

In a further embodiment, the peptide ligand ofC_(i)P[1Nal][dD]C_(ii)M[HArg]DWSTP[HyP]WC_(iii) (SEQ ID NO: 1) comprisesan amino acid sequence selected from:

[B-Ala][Sar10]-(SEQ ID NO: 1) (hereinafter referred to as BCY8234);Ac-[B-Ala][Sar₅]-(SEQ ID NO: 1) (hereinafter referred to as BCY8122);Ac-(SEQ ID NO: 1) (hereinafter referred to as BCY8126);(SEQ ID NO: 1) (hereinafter referred to as BCY8116); andFluorescein-(SEQ ID NO: 1) (hereinafter referred to as BCY8205).

In a yet further embodiment, the peptide ligand ofC_(i)P[1Nal][dD]C_(ii)M[HArg]DWSTP[HyP]WC_(iii) (SEQ ID NO: 1) comprisesan amino acid sequence selected from:

Ac-[B-Ala][Sar₅]-(SEQ ID NO: 1) (hereinafter referred to as BCY8122);Ac-(SEQ ID NO: 1) (hereinafter referred to as BCY8126); and(SEQ ID NO: 1) (hereinafter referred to as BCY8116).

Data is presented herein in Table 2 which demonstrates that the peptideligands of this embodiment exhibited excellent levels of binding tohuman Nectin-4 as evidenced by the SPR binding data.

In a yet further embodiment, the peptide ligand ofC_(i)P[1Nal][dD]C_(ii)M[HArg]DWSTP[HyP]WC_(iii) (SEQ ID NO: 1) comprisesan amino acid sequence selected from:

Ac-[B-Ala][Sar₅]-(SEQ ID NO: 1) (hereinafter referred to as BCY8122);and Ac-(SEQ ID NO: 1) (hereinafter referred to as BCY8126).

Data is presented herein in Table 1 which demonstrates that the peptideligands of this embodiment exhibited excellent levels of binding tohuman Nectin-4 as evidenced by the competition binding data bindingdata.

In a yet further embodiment, the peptide ligand ofC_(i)P[1Nal][dD]C_(ii)M[HArg]DWSTP[HyP]WC_(iii) (SEQ ID NO: 1) comprisesan amino acid sequence selected from:

[B-Ala][Sar10]-(SEQ ID NO: 1) (hereinafter referred to as BCY8234).

Data is presented herein in Table 3 which demonstrates that the peptideligands of this embodiment when conjugated to a cytotoxic agentexhibited excellent levels (<10 nM) of binding to human Nectin-4 asevidenced by the SPR binding data.

In one embodiment, the molecular scaffold is1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA).

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art, such as in the arts of peptide chemistry, cell culture andphage display, nucleic acid chemistry and biochemistry. Standardtechniques are used for molecular biology, genetic and biochemicalmethods (see Sambrook et al., Molecular Cloning: A Laboratory Manual,3rd ed., 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.; Ausubel et al., Short Protocols in Molecular Biology (1999) 4^(th)ed., John Wiley & Sons, Inc.), which are incorporated herein byreference.

Nomenclature

Numbering

When referring to amino acid residue positions within the peptides ofthe invention, cysteine residues (C_(i), C_(ii) and C_(iii)) are omittedfrom the numbering as they are invariant, therefore, the numbering ofamino acid residues within the peptides of the invention is referred toas below:

(SEQ ID NO: 1) C_(i)-P₁-[Nal]₂-[dD]₃-C_(ii)-M₄-[HArg]₅-D₆-W₇-S₈-T₉-P₁₀-[HyP]₁₁-W₁₂-C_(iii)

For the purpose of this description, all bicyclic peptides are assumedto be cyclised with1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)triprop-2-an-1-one (TATA) andyielding a tri-substituted structure. Cyclisation with TATA occurs onC_(i), C_(ii), and C_(iii).

Molecular Format

N- or C-terminal extensions to the bicycle core sequence are added tothe left or right side of the sequence, separated by a hyphen. Forexample, an N-terminal βAla-Sar10-Ala tail would be denoted as:

βAla-Sar10-A-(SEQ ID NO: X).Inversed Peptide Sequences

In light of the disclosure in Nair et al (2003) J Immunol 170(3),1362-1373, it is envisaged that the peptide sequences disclosed hereinwould also find utility in their retro-inverso form. For example, thesequence is reversed (i.e. N-terminus becomes C-terminus and vice versa)and their stereochemistry is likewise also reversed (i.e. D-amino acidsbecome L-amino acids and vice versa).

Peptide Ligands

A peptide ligand, as referred to herein, refers to a peptide covalentlybound to a molecular scaffold. Typically, such peptides comprise two ormore reactive groups (i.e. cysteine residues) which are capable offorming covalent bonds to the scaffold, and a sequence subtended betweensaid reactive groups which is referred to as the loop sequence, since itforms a loop when the peptide is bound to the scaffold. In the presentcase, the peptides comprise at least three cysteine residues (referredto herein as C_(i), C_(ii) and C_(iii)), and form at least two loops onthe scaffold.

Advantages of the Peptide Ligands

Certain bicyclic peptides of the present invention have a number ofadvantageous properties which enable them to be considered as suitabledrug-like molecules for injection, inhalation, nasal, ocular, oral ortopical administration. Such advantageous properties include:

-   -   Species cross-reactivity. This is a typical requirement for        preclinical pharmacodynamics and pharmacokinetic evaluation;    -   Protease stability. Bicyclic peptide ligands should ideally        demonstrate stability to plasma proteases, epithelial        (“membrane-anchored”) proteases, gastric and intestinal        proteases, lung surface proteases, intracellular proteases and        the like. Protease stability should be maintained between        different species such that a bicycle lead candidate can be        developed in animal models as well as administered with        confidence to humans;    -   Desirable solubility profile. This is a function of the        proportion of charged and hydrophilic versus hydrophobic        residues and intra/inter-molecular H-bonding, which is important        for formulation and absorption purposes;    -   An optimal plasma half-life in the circulation. Depending upon        the clinical indication and treatment regimen, it may be        required to develop a bicyclic peptide for short exposure in an        acute illness management setting, or develop a bicyclic peptide        with enhanced retention in the circulation, and is therefore        optimal for the management of more chronic disease states. Other        factors driving the desirable plasma half-life are requirements        of sustained exposure for maximal therapeutic efficiency versus        the accompanying toxicology due to sustained exposure of the        agent; and    -   Selectivity. Certain peptide ligands of the invention        demonstrate good selectivity over other nectins.        Pharmaceutically Acceptable Salts

It will be appreciated that salt forms are within the scope of thisinvention, and references to peptide ligands include the salt forms ofsaid ligands.

The salts of the present invention can be synthesized from the parentcompound that contains a basic or acidic moiety by conventional chemicalmethods such as methods described in Pharmaceutical Salts: Properties,Selection, and Use, P. Heinrich Stahl (Editor), Camille G. Wermuth(Editor), ISBN: 3-90639-026-8, Hardcover, 388 pages, August 2002.Generally, such salts can be prepared by reacting the free acid or baseforms of these compounds with the appropriate base or acid in water orin an organic solvent, or in a mixture of the two.

Acid addition salts (mono- or di-salts) may be formed with a widevariety of acids, both inorganic and organic. Examples of acid additionsalts include mono- or di-salts formed with an acid selected from thegroup consisting of acetic, 2,2-dichloroacetic, adipic, alginic,ascorbic (e.g. L-ascorbic), L-aspartic, benzenesulfonic, benzoic,4-acetamidobenzoic, butanoic, (+) camphoric, camphor-sulfonic,(+)-(1S)-camphor-10-sulfonic, capric, caproic, caprylic, cinnamic,citric, cyclamic, dodecylsulfuric, ethane-1,2-disulfonic,ethanesulfonic, 2-hydroxyethanesulfonic, formic, fumaric, galactaric,gentisic, glucoheptonic, D-gluconic, glucuronic (e.g. D-glucuronic),glutamic (e.g. L-glutamic), α-oxoglutaric, glycolic, hippuric,hydrohalic acids (e.g. hydrobromic, hydrochloric, hydriodic),isethionic, lactic (e.g. (+)-L-lactic, (±)-DL-lactic), lactobionic,maleic, malic, (−)-L-malic, malonic, (±)-DL-mandelic, methanesulfonic,naphthalene-2-sulfonic, naphthalene-1,5-disulfonic,1-hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic,palmitic, pamoic, phosphoric, propionic, pyruvic, L-pyroglutamic,salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulfuric,tannic, (+)-L-tartaric, thiocyanic, p-toluenesulfonic, undecylenic andvaleric acids, as well as acylated amino acids and cation exchangeresins.

One particular group of salts consists of salts formed from acetic,hydrochloric, hydriodic, phosphoric, nitric, sulfuric, citric, lactic,succinic, maleic, malic, isethionic, fumaric, benzenesulfonic,toluenesulfonic, sulfuric, methanesulfonic (mesylate), ethanesulfonic,naphthalenesulfonic, valeric, propanoic, butanoic, malonic, glucuronicand lactobionic acids. One particular salt is the hydrochloride salt.Another particular salt is the acetate salt.

If the compound is anionic, or has a functional group which may beanionic (e.g., —COOH may be —COO⁻), then a salt may be formed with anorganic or inorganic base, generating a suitable cation. Examples ofsuitable inorganic cations include, but are not limited to, alkali metalions such as Li⁺, Na⁺ and K⁺, alkaline earth metal cations such as Ca²⁺and Mg²⁺, and other cations such as Al³⁺ or Zn⁺. Examples of suitableorganic cations include, but are not limited to, ammonium ion (i.e., NH₄⁺) and substituted ammonium ions (e.g., NH₃R⁺, NH₂R₂ ⁺, NHR₃ ⁺, NR₄ ⁺).Examples of some suitable substituted ammonium ions are those derivedfrom: methylamine, ethylamine, diethylamine, propylamine,dicyclohexylamine, triethylamine, butylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine, benzylamine,phenylbenzylamine, choline, meglumine, and tromethamine, as well asamino acids, such as lysine and arginine. An example of a commonquaternary ammonium ion is N(CH₃)₄ ⁺.

Where the peptides of the invention contain an amine function, these mayform quaternary ammonium salts, for example by reaction with analkylating agent according to methods well known to the skilled person.Such quaternary ammonium compounds are within the scope of theinvention.

Modified Derivatives

It will be appreciated that modified derivatives of the peptide ligandsas defined herein are within the scope of the present invention.Examples of such suitable modified derivatives include one or moremodifications selected from: N-terminal and/or C-terminal modifications;replacement of one or more amino acid residues with one or morenon-natural amino acid residues (such as replacement of one or morepolar amino acid residues with one or more isosteric or isoelectronicamino acids; replacement of one or more non-polar amino acid residueswith other non-natural isosteric or isoelectronic amino acids); additionof a spacer group; replacement of one or more oxidation sensitive aminoacid residues with one or more oxidation resistant amino acid residues;replacement of one or more amino acid residues with an alanine,replacement of one or more L-amino acid residues with one or moreD-amino acid residues; N-alkylation of one or more amide bonds withinthe bicyclic peptide ligand; replacement of one or more peptide bondswith a surrogate bond; peptide backbone length modification;substitution of the hydrogen on the alpha-carbon of one or more aminoacid residues with another chemical group, modification of amino acidssuch as cysteine, lysine, glutamate/aspartate and tyrosine with suitableamine, thiol, carboxylic acid and phenol-reactive reagents so as tofunctionalise said amino acids, and introduction or replacement of aminoacids that introduce orthogonal reactivities that are suitable forfunctionalisation, for example azide or alkyne-group bearing amino acidsthat allow functionalisation with alkyne or azide-bearing moieties,respectively.

In one embodiment, the modified derivative comprises an N-terminaland/or C-terminal modification. In a further embodiment, wherein themodified derivative comprises an N-terminal modification using suitableamino-reactive chemistry, and/or C-terminal modification using suitablecarboxy-reactive chemistry. In a further embodiment, said N-terminal orC-terminal modification comprises addition of an effector group,including but not limited to a cytotoxic agent, a radiochelator or achromophore.

In a further embodiment, the modified derivative comprises an N-terminalmodification. In a further embodiment, the N-terminal modificationcomprises an N-terminal acetyl group. In this embodiment, the N-terminalcysteine group (the group referred to herein as C_(i)) is capped withacetic anhydride or other appropriate reagents during peptide synthesisleading to a molecule which is N-terminally acetylated. This embodimentprovides the advantage of removing a potential recognition point foraminopeptidases and avoids the potential for degradation of the bicyclicpeptide.

In an alternative embodiment, the N-terminal modification comprises theaddition of a molecular spacer group which facilitates the conjugationof effector groups and retention of potency of the bicyclic peptide toits target.

In a further embodiment, the modified derivative comprises a C-terminalmodification. In a further embodiment, the C-terminal modificationcomprises an amide group. In this embodiment, the C-terminal cysteinegroup (the group referred to herein as C_(iii)) is synthesized as anamide during peptide synthesis leading to a molecule which isC-terminally amidated. This embodiment provides the advantage ofremoving a potential recognition point for carboxypeptidase and reducesthe potential for proteolytic degradation of the bicyclic peptide.

In one embodiment, the modified derivative comprises replacement of oneor more amino acid residues with one or more non-natural amino acidresidues. In this embodiment, non-natural amino acids may be selectedhaving isosteric/isoelectronic side chains which are neither recognisedby degradative proteases nor have any adverse effect upon targetpotency.

Alternatively, non-natural amino acids may be used having constrainedamino acid side chains, such that proteolytic hydrolysis of the nearbypeptide bond is conformationally and sterically impeded. In particular,these concern proline analogues, bulky sidechains, Cα-disubstitutedderivatives (for example, aminoisobutyric acid, Aib), and cyclo aminoacids, a simple derivative being amino-cyclopropylcarboxylic acid.

In one embodiment, the modified derivative comprises the addition of aspacer group. In a further embodiment, the modified derivative comprisesthe addition of a spacer group to the N-terminal cysteine (C_(i)) and/orthe C-terminal cysteine (C_(iii)).

In one embodiment, the modified derivative comprises replacement of oneor more oxidation sensitive amino acid residues with one or moreoxidation resistant amino acid residues.

In one embodiment, the modified derivative comprises replacement of oneor more charged amino acid residues with one or more hydrophobic aminoacid residues. In an alternative embodiment, the modified derivativecomprises replacement of one or more hydrophobic amino acid residueswith one or more charged amino acid residues. The correct balance ofcharged versus hydrophobic amino acid residues is an importantcharacteristic of the bicyclic peptide ligands. For example, hydrophobicamino acid residues influence the degree of plasma protein binding andthus the concentration of the free available fraction in plasma, whilecharged amino acid residues (in particular arginine) may influence theinteraction of the peptide with the phospholipid membranes on cellsurfaces. The two in combination may influence half-life, volume ofdistribution and exposure of the peptide drug, and can be tailoredaccording to the clinical endpoint. In addition, the correct combinationand number of charged versus hydrophobic amino acid residues may reduceirritation at the injection site (if the peptide drug has beenadministered subcutaneously).

In one embodiment, the modified derivative comprises replacement of oneor more L-amino acid residues with one or more D-amino acid residues.This embodiment is believed to increase proteolytic stability by sterichindrance and by a propensity of D-amino acids to stabilise β-turnconformations (Tugyi et al (2005) PNAS, 102(2), 413-418).

In one embodiment, the modified derivative comprises removal of anyamino acid residues and substitution with alanines. This embodimentprovides the advantage of removing potential proteolytic attack site(s).

It should be noted that each of the above mentioned modifications serveto deliberately improve the potency or stability of the peptide. Furtherpotency improvements based on modifications may be achieved through thefollowing mechanisms:

-   -   Incorporating hydrophobic moieties that exploit the hydrophobic        effect and lead to lower off rates, such that higher affinities        are achieved;    -   Incorporating charged groups that exploit long-range ionic        interactions, leading to faster on rates and to higher        affinities (see for example Schreiber et al, Rapid,        electrostatically assisted association of proteins (1996),        Nature Struct. Biol. 3, 427-31); and    -   Incorporating additional constraint into the peptide, by for        example constraining side chains of amino acids correctly such        that loss in entropy is minimal upon target binding,        constraining the torsional angles of the backbone such that loss        in entropy is minimal upon target binding and introducing        additional cyclisations in the molecule for identical reasons.        (for reviews see Gentilucci et al, Curr. Pharmaceutical Design,        (2010), 16, 3185-203, and Nestor et al, Curr. Medicinal Chem        (2009), 16, 4399-418).        Isotopic Variations

The present invention includes all pharmaceutically acceptable(radio)isotope-labeled peptide ligands of the invention, wherein one ormore atoms are replaced by atoms having the same atomic number, but anatomic mass or mass number different from the atomic mass or mass numberusually found in nature, and peptide ligands of the invention, whereinmetal chelating groups are attached (termed “effector”) that are capableof holding relevant (radio)isotopes, and peptide ligands of theinvention, wherein certain functional groups are covalently replacedwith relevant (radio)isotopes or isotopically labelled functionalgroups.

Examples of isotopes suitable for inclusion in the peptide ligands ofthe invention comprise isotopes of hydrogen, such as ²H (D) and ³H (T),carbon, such as ¹¹C, ¹³C and ¹⁴C, chlorine, such as ³⁶Cl, fluorine, suchas ¹⁸F, iodine, such as ¹²³I, ¹²⁵I and ¹³¹I, nitrogen, such as ¹³N and¹⁵N, oxygen, such as ¹⁵O, ¹⁷O and ¹⁸O, phosphorus, such as ³²P, sulfur,such as ³⁵S, copper, such as ⁶⁴Cu, gallium, such as ⁶⁷Ga or ⁶⁸Ga,yttrium, such as ⁹⁰Y and lutetium, such as ¹⁷⁷Lu, and Bismuth, such as²¹³Bi.

Certain isotopically-labelled peptide ligands of the invention, forexample, those incorporating a radioactive isotope, are useful in drugand/or substrate tissue distribution studies, and to clinically assessthe presence and/or absence of the Nectin-4 target on diseased tissues.The peptide ligands of the invention can further have valuablediagnostic properties in that they can be used for detecting oridentifying the formation of a complex between a labelled compound andother molecules, peptides, proteins, enzymes or receptors. The detectingor identifying methods can use compounds that are labelled withlabelling agents such as radioisotopes, enzymes, fluorescent substances,luminous substances (for example, luminol, luminol derivatives,luciferin, aequorin and luciferase), etc. The radioactive isotopestritium, i.e. ³H (T), and carbon-14, i.e. ¹⁴C, are particularly usefulfor this purpose in view of their ease of incorporation and ready meansof detection.

Substitution with heavier isotopes such as deuterium, i.e. ²H (D), mayafford certain therapeutic advantages resulting from greater metabolicstability, for example, increased in vivo half-life or reduced dosagerequirements, and hence may be preferred in some circumstances.

Substitution with positron emitting isotopes, such as ¹¹C, ¹⁸F, ¹⁵O and¹³N, can be useful in Positron Emission Topography (PET) studies forexamining target occupancy.

Isotopically-labeled compounds of peptide ligands of the invention cangenerally be prepared by conventional techniques known to those skilledin the art or by processes analogous to those described in theaccompanying Examples using an appropriate isotopically-labeled reagentin place of the non-labeled reagent previously employed.

Molecular Scaffold

In one embodiment, the molecular scaffold comprises a non-aromaticmolecular scaffold. References herein to “non-aromatic molecularscaffold” refer to any molecular scaffold as defined herein which doesnot contain an aromatic (i.e. unsaturated) carbocyclic or heterocyclicring system.

Suitable examples of non-aromatic molecular scaffolds are described inHeinis et al (2014) Angewandte Chemie, International Edition 53(6)1602-1606.

As noted in the foregoing documents, the molecular scaffold may be asmall molecule, such as a small organic molecule.

In one embodiment the molecular scaffold may be a macromolecule. In oneembodiment the molecular scaffold is a macromolecule composed of aminoacids, nucleotides or carbohydrates.

In one embodiment the molecular scaffold comprises reactive groups thatare capable of reacting with functional group(s) of the polypeptide toform covalent bonds.

The molecular scaffold may comprise chemical groups which form thelinkage with a peptide, such as amines, thiols, alcohols, ketones,aldehydes, nitriles, carboxylic acids, esters, alkenes, alkynes, azides,anhydrides, succinimides, maleimides, alkyl halides and acyl halides.

An example of an αβ unsaturated carbonyl containing compound is1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA)(Angewandte Chemie, International Edition (2014), 53(6), 1602-1606).

Effector and Functional Groups

According to a further aspect of the invention, there is provided a drugconjugate comprising a peptide ligand as defined herein conjugated toone or more effector and/or functional groups.

Effector and/or functional groups can be attached, for example, to the Nand/or C termini of the polypeptide, to an amino acid within thepolypeptide, or to the molecular scaffold.

Appropriate effector groups include antibodies and parts or fragmentsthereof. For instance, an effector group can include an antibody lightchain constant region (CL), an antibody CH1 heavy chain domain, anantibody CH2 heavy chain domain, an antibody CH3 heavy chain domain, orany combination thereof, in addition to the one or more constant regiondomains. An effector group may also comprise a hinge region of anantibody (such a region normally being found between the CH1 and CH2domains of an IgG molecule).

In a further embodiment of this aspect of the invention, an effectorgroup according to the present invention is an Fc region of an IgGmolecule. Advantageously, a peptide ligand-effector group according tothe present invention comprises or consists of a peptide ligand Fcfusion having a tβ half-life of a day or more, two days or more, 3 daysor more, 4 days or more, 5 days or more, 6 days or more or 7 days ormore. Most advantageously, the peptide ligand according to the presentinvention comprises or consists of a peptide ligand Fc fusion having atβ half-life of a day or more.

Functional groups include, in general, binding groups, drugs, reactivegroups for the attachment of other entities, functional groups which aiduptake of the macrocyclic peptides into cells, and the like.

The ability of peptides to penetrate into cells will allow peptidesagainst intracellular targets to be effective. Targets that can beaccessed by peptides with the ability to penetrate into cells includetranscription factors, intracellular signalling molecules such astyrosine kinases and molecules involved in the apoptotic pathway.Functional groups which enable the penetration of cells include peptidesor chemical groups which have been added either to the peptide or themolecular scaffold. Peptides such as those derived from such as VP22,HIV-Tat, a homeobox protein of Drosophila (Antennapedia), e.g. asdescribed in Chen and Harrison, Biochemical Society Transactions (2007)Volume 35, part 4, p 821; Gupta et al. in Advanced Drug DiscoveryReviews (2004) Volume 57 9637. Examples of short peptides which havebeen shown to be efficient at translocation through plasma membranesinclude the 16 amino acid penetratin peptide from DrosophilaAntennapedia protein (Derossi et al (1994) J Biol. Chem. Volume 269 p10444), the 18 amino acid ‘model amphipathic peptide’ (Oehlke et al(1998) Biochim Biophys Acts Volume 1414 p 127) and arginine rich regionsof the HIV TAT protein. Non peptidic approaches include the use of smallmolecule mimics or SMOCs that can be easily attached to biomolecules(Okuyama et al (2007) Nature Methods Volume 4 p 153). Other chemicalstrategies to add guanidinium groups to molecules also enhance cellpenetration (Elson-Scwab et al (2007) J Biol Chem Volume 282 p 13585).Small molecular weight molecules such as steroids may be added to themolecular scaffold to enhance uptake into cells.

One class of functional groups which may be attached to peptide ligandsincludes antibodies and binding fragments thereof, such as Fab, Fv orsingle domain fragments. In particular, antibodies which bind toproteins capable of increasing the half-life of the peptide ligand invivo may be used.

In one embodiment, a peptide ligand-effector group according to theinvention has a tβ half-life selected from the group consisting of: 12hours or more, 24 hours or more, 2 days or more, 3 days or more, 4 daysor more, 5 days or more, 6 days or more, 7 days or more, 8 days or more,9 days or more, 10 days or more, 11 days or more, 12 days or more, 13days or more, 14 days or more, 15 days or more or 20 days or more.Advantageously a peptide ligand-effector group or composition accordingto the invention will have a tβ half-life in the range 12 to 60 hours.In a further embodiment, it will have a tβ half-life of a day or more.In a further embodiment still, it will be in the range 12 to 26 hours.

In one particular embodiment of the invention, the functional group isselected from a metal chelator, which is suitable for complexing metalradioisotopes of medicinal relevance.

Possible effector groups also include enzymes, for instance such ascarboxypeptidase G2 for use in enzyme/prodrug therapy, where the peptideligand replaces antibodies in ADEPT.

In one particular embodiment of the invention, the functional group isselected from a drug, such as a cytotoxic agent for cancer therapy.Suitable examples include: alkylating agents such as cisplatin andcarboplatin, as well as oxaliplatin, mechlorethamine, cyclophosphamide,chlorambucil, ifosfamide; Anti-metabolites including purine analogsazathioprine and mercaptopurine or pyrimidine analogs; plant alkaloidsand terpenoids including vinca alkaloids such as Vincristine,Vinblastine, Vinorelbine and Vindesine; Podophyllotoxin and itsderivatives etoposide and teniposide; Taxanes, including paclitaxel,originally known as Taxol; topoisomerase inhibitors includingcamptothecins: irinotecan and topotecan, and type II inhibitorsincluding amsacrine, etoposide, etoposide phosphate, and teniposide.Further agents can include antitumour antibiotics which include theimmunosuppressant dactinomycin (which is used in kidneytransplantations), doxorubicin, epirubicin, bleomycin, calicheamycins,and others.

In one further particular embodiment of the invention, the cytotoxicagent is selected from maytansinoids (such as DM1) or monomethylauristatins (such as MMAE).

DM1 is a cytotoxic agent which is a thiol-containing derivative ofmaytansine and has the following structure:

Data is presented herein in Table 3 which demonstrates the effects of apeptide ligand conjugated to a toxin containing DM1.

Monomethyl auristatin E (MMAE) is a synthetic antineoplastic agent andhas the following structure:

Data is presented herein in Table 3 which demonstrates the effects ofpeptide ligands conjugated to a toxin containing MMAE.

In one yet further particular embodiment of the invention, the cytotoxicagent is selected from monomethyl auristatin E (MMAE).

In one embodiment, the cytotoxic agent is linked to the bicyclic peptideby a cleavable bond, such as a disulphide bond or a protease sensitivebond. In a further embodiment, the groups adjacent to the disulphidebond are modified to control the hindrance of the disulphide bond, andby this the rate of cleavage and concomitant release of cytotoxic agent.

Published work established the potential for modifying thesusceptibility of the disulphide bond to reduction by introducing sterichindrance on either side of the disulphide bond (Kellogg et al (2011)Bioconjugate Chemistry, 22, 717). A greater degree of steric hindrancereduces the rate of reduction by intracellular glutathione and alsoextracellular (systemic) reducing agents, consequentially reducing theease by which toxin is released, both inside and outside the cell. Thus,selection of the optimum in disulphide stability in the circulation(which minimises undesirable side effects of the toxin) versus efficientrelease in the intracellular milieu (which maximises the therapeuticeffect) can be achieved by careful selection of the degree of hindranceon either side of the disulphide bond.

The hindrance on either side of the disulphide bond is modulated throughintroducing one or more methyl groups on either the targeting entity(here, the bicyclic peptide) or toxin side of the molecular construct.

In one embodiment, the cytotoxic agent and linker is selected from anycombinations of those described in WO 2016/067035 (the cytotoxic agentsand linkers thereof are herein incorporated by reference).

In one embodiment, the linker between said cytotoxic agent and saidbicyclic peptide comprises one or more amino acid residues. Examples ofsuitable amino acid residues as suitable linkers include Ala, Cit, Lys,Trp and Val.

In one embodiment, the cytotoxic agent is selected from MMAE and saiddrug conjugate additionally comprises a linker selected from:-PABC-Cit-Val-Glutaryl- or -PABC-cyclobutyl-Ala-Cit-βAla-, wherein PABCrepresents p-aminobenzylcarbamate. Full details of the cyclobutylcontaining linker may be found in Wei et al (2018) J. Med. Chem. 61,989-1000. In a further embodiment, the cytotoxic agent is selected fromMMAE and the linker is -PABC-Cit-Val-Glutaryl-.

In an alternative embodiment, the cytotoxic agent is DM1 and said drugconjugate additionally comprises a linker which is -SPDB-(SO₃H)—,wherein SPDB represents N-succinimidyl 3-(2-pyridyldithio)propionate.

In an alternative embodiment, the cytotoxic agent is MMAE, the bicyclicpeptide is selected from BCY8234 as defined herein and the linker isselected from -PABC-Cit-Val-Glutaryl-. This BDC is known herein asBCY8245 and is

represented in a more detailed manner as:

Data is presented herein which demonstrates excellent binding to humanNectin-4 for BCY8245 in the SPR binding assay as shown in Table 3. ThisBDC also demonstrated good anti-tumour activity in the Non-Small CellLung Cancer model as shown in Example 1, the bladder cancer model asshown in Example 2, the pancreatic cancer model as shown in Example 3and the breast cancer model as shown in Example 4.

In an alternative embodiment, the cytotoxic agent is MMAE, the bicyclicpeptide is selected from BCY8234 as defined herein and the linker isselected from -PABC-cyclobutyl-(B-Ala)-. This BDC is known herein asBCY8549. Data is presented herein which demonstrates excellent bindingto human Nectin-4 for BCY8549 in the SPR binding assay as shown in Table3.

In a further embodiment, the bicyclic drug conjugate is selected fromBCY8245 or BCY8549. In a yet further embodiment, the bicyclic drugconjugate is BCY8245. The drug conjugate BCY8245 demonstrated superiordose dependent anti-tumour activity as demonstrated in the datadescribed herein.

Synthesis

The peptides of the present invention may be manufactured syntheticallyby standard techniques followed by reaction with a molecular scaffold invitro. When this is performed, standard chemistry may be used. Thisenables the rapid large scale preparation of soluble material forfurther downstream experiments or validation. Such methods could beaccomplished using conventional chemistry such as that disclosed inTimmerman et al (supra).

Thus, the invention also relates to manufacture of polypeptides orconjugates selected as set out herein, wherein the manufacture comprisesoptional further steps as explained below. In one embodiment, thesesteps are carried out on the end product polypeptide/conjugate made bychemical synthesis.

Optionally amino acid residues in the polypeptide of interest may besubstituted when manufacturing a conjugate or complex.

Peptides can also be extended, to incorporate for example another loopand therefore introduce multiple specificities.

To extend the peptide, it may simply be extended chemically at itsN-terminus or C-terminus or within the loops using orthogonallyprotected lysines (and analogues) using standard solid phase or solutionphase chemistry. Standard (bio)conjugation techniques may be used tointroduce an activated or activatable N- or C-terminus. Alternativelyadditions may be made by fragment condensation or native chemicalligation e.g. as described in (Dawson et al. 1994. Synthesis of Proteinsby Native Chemical Ligation. Science 266:776-779), or by enzymes, forexample using subtiligase as described in (Chang et al. Proc Natl AcadSci USA. 1994 Dec. 20; 91(26):12544-8 or in Hikari et al Bioorganic &Medicinal Chemistry Letters Volume 18, Issue 22, 15 Nov. 2008, Pages6000-6003).

Alternatively, the peptides may be extended or modified by furtherconjugation through disulphide bonds. This has the additional advantageof allowing the first and second peptide to dissociate from each otheronce within the reducing environment of the cell. In this case, themolecular scaffold (e.g. TATA) could be added during the chemicalsynthesis of the first peptide so as to react with the three cysteinegroups; a further cysteine or thiol could then be appended to the N- orC-terminus of the first peptide, so that this cysteine or thiol onlyreacted with a free cysteine or thiol of the second peptide, forming adisulfide-linked bicyclic peptide-peptide conjugate.

Similar techniques apply equally to the synthesis/coupling of twobicyclic and bispecific macrocycles, potentially creating atetraspecific molecule.

Furthermore, addition of other functional groups or effector groups maybe accomplished in the same manner, using appropriate chemistry,coupling at the N- or C-termini or via side chains. In one embodiment,the coupling is conducted in such a manner that it does not block theactivity of either entity.

Pharmaceutical Compositions

According to a further aspect of the invention, there is provided apharmaceutical composition comprising a peptide ligand or a drugconjugate as defined herein in combination with one or morepharmaceutically acceptable excipients.

Generally, the present peptide ligands will be utilised in purified formtogether with pharmacologically appropriate excipients or carriers.Typically, these excipients or carriers include aqueous oralcoholic/aqueous solutions, emulsions or suspensions, including salineand/or buffered media. Parenteral vehicles include sodium chloridesolution, Ringer's dextrose, dextrose and sodium chloride and lactatedRinger's. Suitable physiologically-acceptable adjuvants, if necessary tokeep a polypeptide complex in suspension, may be chosen from thickenerssuch as carboxymethylcellulose, polyvinylpyrrolidone, gelatin andalginates.

Intravenous vehicles include fluid and nutrient replenishers andelectrolyte replenishers, such as those based on Ringer's dextrose.Preservatives and other additives, such as antimicrobials, antioxidants,chelating agents and inert gases, may also be present (Mack (1982)Remington's Pharmaceutical Sciences, 16th Edition).

The peptide ligands of the present invention may be used as separatelyadministered compositions or in conjunction with other agents. These caninclude antibodies, antibody fragments and various immunotherapeuticdrugs, such as cylcosporine, methotrexate, adriamycin or cisplatinum andimmunotoxins. Pharmaceutical compositions can include “cocktails” ofvarious cytotoxic or other agents in conjunction with the proteinligands of the present invention, or even combinations of selectedpolypeptides according to the present invention having differentspecificities, such as polypeptides selected using different targetligands, whether or not they are pooled prior to administration.

The route of administration of pharmaceutical compositions according tothe invention may be any of those commonly known to those of ordinaryskill in the art. For therapy, the peptide ligands of the invention canbe administered to any patient in accordance with standard techniques.The administration can be by any appropriate mode, includingparenterally, intravenously, intramuscularly, intraperitoneally,transdermally, via the pulmonary route, or also, appropriately, bydirect infusion with a catheter. Preferably, the pharmaceuticalcompositions according to the invention will be administered byinhalation. The dosage and frequency of administration will depend onthe age, sex and condition of the patient, concurrent administration ofother drugs, counterindications and other parameters to be taken intoaccount by the clinician.

The peptide ligands of this invention can be lyophilised for storage andreconstituted in a suitable carrier prior to use. This technique hasbeen shown to be effective and art-known lyophilisation andreconstitution techniques can be employed. It will be appreciated bythose skilled in the art that lyophilisation and reconstitution can leadto varying degrees of activity loss and that levels may have to beadjusted upward to compensate.

The compositions containing the present peptide ligands or a cocktailthereof can be administered for prophylactic and/or therapeutictreatments. In certain therapeutic applications, an adequate amount toaccomplish at least partial inhibition, suppression, modulation,killing, or some other measurable parameter, of a population of selectedcells is defined as a “therapeutically-effective dose”. Amounts neededto achieve this dosage will depend upon the severity of the disease andthe general state of the patient's own immune system, but generallyrange from 0.005 to 5.0 mg of selected peptide ligand per kilogram ofbody weight, with doses of 0.05 to 2.0 mg/kg/dose being more commonlyused. For prophylactic applications, compositions containing the presentpeptide ligands or cocktails thereof may also be administered in similaror slightly lower dosages.

A composition containing a peptide ligand according to the presentinvention may be utilised in prophylactic and therapeutic settings toaid in the alteration, inactivation, killing or removal of a selecttarget cell population in a mammal. In addition, the peptide ligandsdescribed herein may be used extracorporeally or in vitro selectively tokill, deplete or otherwise effectively remove a target cell populationfrom a heterogeneous collection of cells. Blood from a mammal may becombined extracorporeally with the selected peptide ligands whereby theundesired cells are killed or otherwise removed from the blood forreturn to the mammal in accordance with standard techniques.

Co-Administration with One or More Other Therapeutic Agent

Depending upon the particular condition, or disease, to be treated,additional therapeutic agents that are normally administered to treatthat condition, may also be present in the compositions of thisinvention. Thus, in one embodiment, the pharmaceutical compositionadditionally comprises one or more therapeutic agents. As used herein,additional therapeutic agents that are normally administered to treat aparticular disease, or condition, are known as “appropriate for thedisease, or condition, being treated.”

In some embodiments, the present invention provides a method of treatinga disclosed disease or condition comprising administering to a patientin need thereof an effective amount of a compound disclosed herein or apharmaceutically acceptable salt thereof and co-administeringsimultaneously or sequentially an effective amount of one or moreadditional therapeutic agents, such as those described herein. In someembodiments, the method includes co-administering one additionaltherapeutic agent. In some embodiments, the method includesco-administering two additional therapeutic agents. In some embodiments,the combination of the disclosed compound and the additional therapeuticagent or agents acts synergistically.

A compound of the current invention may also be used in combination withknown therapeutic processes, for example, the administration of hormonesor radiation. In certain embodiments, a provided compound is used as aradiosensitizer, especially for the treatment of tumors which exhibitpoor sensitivity to radiotherapy.

A compound of the current invention can be administered alone or incombination with one or more other therapeutic compounds, possiblecombination therapy taking the form of fixed combinations or theadministration of a compound of the invention and one or more othertherapeutic compounds being staggered or given independently of oneanother, or the combined administration of fixed combinations and one ormore other therapeutic compounds. A compound of the current inventioncan besides or in addition be administered especially for tumor therapyin combination with chemotherapy, radiotherapy, immunotherapy,phototherapy, surgical intervention, or a combination of these.Long-term therapy is equally possible as is adjuvant therapy in thecontext of other treatment strategies, as described above. Otherpossible treatments are therapy to maintain the patient's status aftertumor regression, or even chemopreventive therapy, for example inpatients at risk.

One or more other therapeutic agent may be administered separately froma compound or composition of the invention, as part of a multiple dosageregimen. Alternatively, one or more other therapeutic agents may be partof a single dosage form, mixed together with a compound of thisinvention in a single composition. If administered as a multiple dosageregime, one or more other therapeutic agent and a compound orcomposition of the invention may be administered simultaneously,sequentially or within a period of time from one another, for examplewithin 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, or 24 hours from one another. In some embodiments,one or more other therapeutic agent and a compound or composition of theinvention are administered as a multiple dosage regimen within greaterthan 24 hours apart.

As used herein, the term “combination,” “combined,” and related termsrefers to the simultaneous or sequential administration of therapeuticagents in accordance with this invention. For example, a compound of thepresent invention may be administered with one or more other therapeuticagent simultaneously or sequentially in separate unit dosage forms ortogether in a single unit dosage form. Accordingly, the presentinvention provides a single unit dosage form comprising a compound ofthe current invention, one or more other therapeutic agent, and apharmaceutically acceptable carrier, adjuvant, or vehicle.

The amount of a compound of the invention and one or more othertherapeutic agent (in those compositions which comprise an additionaltherapeutic agent as described above) that may be combined with thecarrier materials to produce a single dosage form will vary dependingupon the host treated and the particular mode of administration.Preferably, a composition of the invention should be formulated so thata dosage of between 0.01-100 mg/kg body weight/day of a compound of theinvention can be administered.

In those compositions which comprise one or more other therapeuticagent, the one or more other therapeutic agent and a compound of theinvention may act synergistically. Therefore, the amount of the one ormore other therapeutic agent in such compositions may be less than thatrequired in a monotherapy utilizing only that therapeutic agent. In suchcompositions a dosage of between 0.01-1,000 μg/kg body weight/day of theone or more other therapeutic agent can be administered.

The amount of one or more other therapeutic agent present in thecompositions of this invention may be no more than the amount that wouldnormally be administered in a composition comprising that therapeuticagent as the only active agent. Preferably the amount of one or moreother therapeutic agent in the presently disclosed compositions willrange from about 50% to 100% of the amount normally present in acomposition comprising that agent as the only therapeutically activeagent. In some embodiments, one or more other therapeutic agent isadministered at a dosage of about 50%, about 55%, about 60%, about 65%,about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% ofthe amount normally administered for that agent. As used herein, thephrase “normally administered” means the amount of an FDA approvedtherapeutic agent provided for dosing as per the FDA label insert.

The compounds of this invention, or pharmaceutical compositions thereof,may also be incorporated into compositions for coating an implantablemedical device, such as prostheses, artificial valves, vascular grafts,stents and catheters. Vascular stents, for example, have been used toovercome restenosis (re-narrowing of the vessel wall after injury).However, patients using stents or other implantable devices risk clotformation or platelet activation. These unwanted effects may beprevented or mitigated by pre-coating the device with a pharmaceuticallyacceptable composition comprising a kinase inhibitor. Implantabledevices coated with a compound of this invention are another embodimentof the present invention.

Exemplary Other Therapeutic Agents

In some embodiments, one or more other therapeutic agent is a Poly ADPribose polymerase (PARP) inhibitor. In some embodiments, a PARPinhibitor is selected from olaparib (Lynparza®, AstraZeneca); rucaparib(Rubraca®, Clovis Oncology); niraparib (Zejula®, Tesaro); talazoparib(MDV3800/BMN 673/LT00673, Medivation/Pfizer/Biomarin); veliparib(ABT-888, AbbVie); and BGB-290 (BeiGene, Inc.).

In some embodiments, one or more other therapeutic agent is a histonedeacetylase (HDAC) inhibitor. In some embodiments, an HDAC inhibitor isselected from vorinostat (Zolinza®, Merck); romidepsin (Istodax®,Celgene); panobinostat (Farydak®, Novartis); belinostat (Beleodaq®,Spectrum Pharmaceuticals); entinostat (SNDX-275, Syndax Pharmaceuticals)(NCT00866333); and chidamide (Epidaza®, HBI-8000, ChipscreenBiosciences, China).

In some embodiments, one or more other therapeutic agent is a CDKinhibitor, such as a CDK4/CDK6 inhibitor. In some embodiments, a CDK 4/6inhibitor is selected from palbociclib (Ibrance®, Pfizer); ribociclib(Kisqali®, Novartis); abemaciclib (Ly2835219, Eli Lilly); andtrilaciclib (G1T28, G1 Therapeutics).

In some embodiments, one or more other therapeutic agent is aphosphatidylinositol 3 kinase (PI3K) inhibitor. In some embodiments, aPI3K inhibitor is selected from idelalisib (Zydelig®, Gilead), alpelisib(BYL719, Novartis), taselisib (GDC-0032, Genentech/Roche); pictilisib(GDC-0941, Genentech/Roche); copanlisib (BAY806946, Bayer); duvelisib(formerly IPI-145, Infinity Pharmaceuticals); PQR309 (PiqurTherapeutics, Switzerland); and TGR1202 (formerly RP5230, TGTherapeutics).

In some embodiments, one or more other therapeutic agent is aplatinum-based therapeutic, also referred to as platins. Platins causecross-linking of DNA, such that they inhibit DNA repair and/or DNAsynthesis, mostly in rapidly reproducing cells, such as cancer cells. Insome embodiments, a platinum-based therapeutic is selected fromcisplatin (Platinol®, Bristol-Myers Squibb); carboplatin (Paraplatin®,Bristol-Myers Squibb; also, Teva; Pfizer); oxaliplatin (Eloxitin®Sanofi-Aventis); nedaplatin (Aqupla®, Shionogi), picoplatin (PoniardPharmaceuticals); and satraplatin (JM-216, Agennix).

In some embodiments, one or more other therapeutic agent is a taxanecompound, which causes disruption of microtubules, which are essentialfor cell division. In some embodiments, a taxane compound is selectedfrom paclitaxel (Taxol®, Bristol-Myers Squibb), docetaxel (Taxotere®,Sanofi-Aventis; Docefrez®, Sun Pharmaceutical), albumin-bound paclitaxel(Abraxane®; Abraxis/Celgene), cabazitaxel (Jevtana®, Sanofi-Aventis),and SID530 (SK Chemicals, Co.) (NCT00931008).

In some embodiments, one or more other therapeutic agent is a nucleosideinhibitor, or a therapeutic agent that interferes with normal DNAsynthesis, protein synthesis, cell replication, or will otherwiseinhibit rapidly proliferating cells.

In some embodiments, a nucleoside inhibitor is selected from trabectedin(guanidine alkylating agent, Yondelis®, Janssen Oncology),mechlorethamine (alkylating agent, Valchlor®, Aktelion Pharmaceuticals);vincristine (Oncovin®, Eli Lilly; Vincasar®, Teva Pharmaceuticals;Marqibo®, Talon Therapeutics); temozolomide (prodrug to alkylating agent5-(3-methyltriazen-1-yl)-imidazole-4-carboxamide (MTIC) Temodar®,Merck); cytarabine injection (ara-C, antimetabolic cytidine analog,Pfizer); lomustine (alkylating agent, CeeNU®, Bristol-Myers Squibb;Gleostine®, NextSource Biotechnology); azacitidine (pyrimidinenucleoside analog of cytidine, Vidaza®, Celgene); omacetaxinemepesuccinate (cephalotaxine ester) (protein synthesis inhibitor,Synribo®; Teva Pharmaceuticals); asparaginase Erwinia chrysanthemi(enzyme for depletion of asparagine, Elspar®, Lundbeck; Erwinaze®, EUSAPharma); eribulin mesylate (microtubule inhibitor, tubulin-basedantimitotic, Halaven®, Eisai); cabazitaxel (microtubule inhibitor,tubulin-based antimitotic, Jevtana®, Sanofi-Aventis); capacetrine(thymidylate synthase inhibitor, Xeloda®, Genentech); bendamustine(bifunctional mechlorethamine derivative, believed to form interstrandDNA cross-links, Treanda®, Cephalon/Teva); ixabepilone (semi-syntheticanalog of epothilone B, microtubule inhibitor, tubulin-basedantimitotic, Ixempra®, Bristol-Myers Squibb); nelarabine (prodrug ofdeoxyguanosine analog, nucleoside metabolic inhibitor, Arranon®,Novartis); clorafabine (prodrug of ribonucleotide reductase inhibitor,competitive inhibitor of deoxycytidine, Clolar®, Sanofi-Aventis); andtrifluridine and tipiracil (thymidine-based nucleoside analog andthymidine phosphorylase inhibitor, Lonsurf®, Taiho Oncology).

In some embodiments, one or more other therapeutic agent is a kinaseinhibitor or VEGF-R antagonist. Approved VEGF inhibitors and kinaseinhibitors useful in the present invention include: bevacizumab(Avastin®, Genentech/Roche) an anti-VEGF monoclonal antibody;ramucirumab (Cyramza®, Eli Lilly), an anti-VEGFR-2 antibody andziv-aflibercept, also known as VEGF Trap (Zaltrap®; Regeneron/Sanofi).VEGFR inhibitors, such as regorafenib (Stivarga®, Bayer); vandetanib(Caprelsa®, AstraZeneca); axitinib (Inlyta®, Pfizer); and lenvatinib(Lenvima®, Eisai); Raf inhibitors, such as sorafenib (Nexavar®, Bayer AGand Onyx); dabrafenib (Tafinlar®, Novartis); and vemurafenib (Zelboraf®,Genentech/Roche); MEK inhibitors, such as cobimetanib (Cotellic®,Exelexis/Genentech/Roche); trametinib (Mekinist®, Novartis); Bcr-Abltyrosine kinase inhibitors, such as imatinib (Gleevec®, Novartis);nilotinib (Tasigna®, Novartis); dasatinib (Sprycel®,BristolMyersSquibb); bosutinib (Bosulif®, Pfizer); and ponatinib(Inclusig®, Ariad Pharmaceuticals); Her2 and EGFR inhibitors, such asgefitinib (Iressa®, AstraZeneca); erlotinib (Tarceeva®,Genentech/Roche/Astellas); lapatinib (Tykerb®, Novartis); afatinib(Gilotrif®, Boehringer Ingelheim); osimertinib (targeting activatedEGFR, Tagrisso®, AstraZeneca); and brigatinib (Alunbrig®, AriadPharmaceuticals); c-Met and VEGFR2 inhibitors, such as cabozanitib(Cometriq®, Exelexis); and multikinase inhibitors, such as sunitinib(Sutent®, Pfizer); pazopanib (Votrient®, Novartis); ALK inhibitors, suchas crizotinib (Xalkori®, Pfizer); ceritinib (Zykadia®, Novartis); andalectinib (Alecenza®, Genentech/Roche); Bruton's tyrosine kinaseinhibitors, such as ibrutinib (Imbruvica®, Pharmacyclics/Janssen); andFlt3 receptor inhibitors, such as midostaurin (Rydapt®, Novartis).

Other kinase inhibitors and VEGF-R antagonists that are in developmentand may be used in the present invention include: tivozanib (AveoPharmaceuticals); vatalanib (Bayer/Novartis); lucitanib (ClovisOncology); dovitinib (TKI258, Novartis); Chiauanib (ChipscreenBiosciences); CEP-11981 (Cephalon); linifanib (Abbott Laboratories);neratinib (HKI-272, Puma Biotechnology); radotinib (Supect®, IY5511,II-Yang Pharmaceuticals, S. Korea); ruxolitinib (Jakafi®, IncyteCorporation); PTC299 (PTC Therapeutics); CP-547,632 (Pfizer); foretinib(Exelexis, GlaxoSmithKline); quizartinib (Daiichi Sankyo) and motesanib(Amgen/Takeda).

In some embodiments, one or more other therapeutic agent is an mTORinhibitor, which inhibits cell proliferation, angiogenesis and glucoseuptake. In some embodiments, an mTOR inhibitor is everolimus (Afinitor®,Novartis); temsirolimus (Torisel®, Pfizer); and sirolimus (Rapamune®,Pfizer).

In some embodiments, one or more other therapeutic agent is a proteasomeinhibitor. Approved proteasome inhibitors useful in the presentinvention include bortezomib (Velcade®, Takeda); carfilzomib (Kyprolis®,Amgen); and ixazomib (Ninlaro®, Takeda).

In some embodiments, one or more other therapeutic agent is a growthfactor antagonist, such as an antagonist of platelet-derived growthfactor (PDGF), or epidermal growth factor (EGF) or its receptor (EGFR).Approved PDGF antagonists which may be used in the present inventioninclude olaratumab (Lartruvo®; Eli Lilly). Approved EGFR antagonistswhich may be used in the present invention include: cetuximab (Erbitux®,Eli Lilly); necitumumab (Portrazza®, Eli Lilly), panitumumab (Vectibix®,Amgen); and osimertinib (targeting activated EGFR, Tagrisso®,AstraZeneca).

In some embodiments, one or more other therapeutic agent is an aromataseinhibitor. In some embodiments, an aromatase inhibitor is selected from:exemestane (Aromasin®, Pfizer); anastazole (Arimidex®, AstraZeneca) andletrozole (Femara®, Novartis).

In some embodiments, one or more other therapeutic agent is anantagonist of the hedgehog pathway. Approved hedgehog pathway inhibitorswhich may be used in the present invention include: sonidegib (Odomzo®,Sun Pharmaceuticals); and vismodegib (Erivedge®, Genentech), both fortreatment of basal cell carcinoma.

In some embodiments, one or more other therapeutic agent is a folic acidinhibitor. Approved folic acid inhibitors useful in the presentinvention include pemetrexed (Alimta®, Eli Lilly).

In some embodiments, one or more other therapeutic agent is a CCchemokine receptor 4 (CCR4) inhibitor. CCR4 inhibitors being studiedthat may be useful in the present invention include mogamulizumab(Poteligeo®, Kyowa Hakko Kirin, Japan).

In some embodiments, one or more other therapeutic agent is anisocitrate dehydrogenase (IDH) inhibitor. IDH inhibitors being studiedwhich may be used in the present invention include: AG 120 (Celgene;NCT02677922); AG221 (Celgene, NCT02677922; NCT02577406); BAY1436032(Bayer, NCT02746081); IDH305 (Novartis, NCT02987010).

In some embodiments, one or more other therapeutic agent is an arginaseinhibitor. Arginase inhibitors being studied which may be used in thepresent invention include: AEB1102 (pegylated recombinant arginase,Aeglea Biotherapeutics), which is being studied in Phase 1 clinicaltrials for acute myeloid leukemia and myelodysplastic syndrome(NCT02732184) and solid tumors (NCT02561234); and CB-1158 (CalitheraBiosciences).

In some embodiments, one or more other therapeutic agent is aglutaminase inhibitor. Glutaminase inhibitors being studied which may beused in the present invention include CB-839 (Calithera Biosciences).

In some embodiments, one or more other therapeutic agent is an antibodythat binds to tumor antigens, that is, proteins expressed on the cellsurface of tumor cells. Approved antibodies that bind to tumor antigenswhich may be used in the present invention include: rituximab (Rituxan®,Genentech/Biogenldec); ofatumumab (anti-CD20, Arzerra®,GlaxoSmithKline); obinutuzumab (anti-CD20, Gazyva®, Genentech),ibritumomab (anti-CD20 and Yttrium-90, Zevalin®, SpectrumPharmaceuticals); daratumumab (anti-CD38, Darzalex®, Janssen Biotech),dinutuximab (anti-glycolipid GD2, Unituxin®, United Therapeutics);trastuzumab (anti-HER2, Herceptin®, Genentech); ado-trastuzumabemtansine (anti-HER2, fused to emtansine, Kadcyla®, Genentech); andpertuzumab (anti-HER2, Perjeta®, Genentech); and brentuximab vedotin(anti-CD30-drug conjugate, Adcetris®, Seattle Genetics).

In some embodiments, one or more other therapeutic agent is atopoisomerase inhibitor. Approved topoisomerase inhibitors useful in thepresent invention include: irinotecan (Onivyde®, MerrimackPharmaceuticals); topotecan (Hycamtin®, GlaxoSmithKline). Topoisomeraseinhibitors being studied which may be used in the present inventioninclude pixantrone (Pixuvri®, CTI Biopharma).

In some embodiments, one or more other therapeutic agent is an inhibitorof anti-apoptotic proteins, such as BCL-2. Approved anti-apoptoticswhich may be used in the present invention include: venetoclax(Venclexta®, AbbVie/Genentech); and blinatumomab (Blincyto®, Amgen).Other therapeutic agents targeting apoptotic proteins which haveundergone clinical testing and may be used in the present inventioninclude navitoclax (ABT-263, Abbott), a BCL-2 inhibitor (NCT02079740).

In some embodiments, one or more other therapeutic agent is an androgenreceptor inhibitor. Approved androgen receptor inhibitors useful in thepresent invention include enzalutamide (Xtandi®, Astellas/Medivation);approved inhibitors of androgen synthesis include abiraterone (Zytiga®,Centocor/Ortho); approved antagonist of gonadotropin-releasing hormone(GnRH) receptor (degaralix, Firmagon®, Ferring Pharmaceuticals).

In some embodiments, one or more other therapeutic agent is a selectiveestrogen receptor modulator (SERM), which interferes with the synthesisor activity of estrogens. Approved SERMs useful in the present inventioninclude raloxifene (Evista®, Eli Lilly).

In some embodiments, one or more other therapeutic agent is an inhibitorof bone resorption. An approved therapeutic which inhibits boneresorption is Denosumab (Xgeva®, Amgen), an antibody that binds toRANKL, prevents binding to its receptor RANK, found on the surface ofosteoclasts, their precursors, and osteoclast-like giant cells, whichmediates bone pathology in solid tumors with osseous metastases. Otherapproved therapeutics that inhibit bone resorption includebisphosphonates, such as zoledronic acid (Zometa®, Novartis).

In some embodiments, one or more other therapeutic agent is an inhibitorof interaction between the two primary p53 suppressor proteins, MDMX andMDM2. Inhibitors of p53 suppression proteins being studied which may beused in the present invention include ALRN-6924 (Aileron), a stapledpeptide that equipotently binds to and disrupts the interaction of MDMXand MDM2 with p53. ALRN-6924 is currently being evaluated in clinicaltrials for the treatment of AML, advanced myelodysplastic syndrome (MDS)and peripheral T-cell lymphoma (PTCL) (NCT02909972; NCT02264613).

In some embodiments, one or more other therapeutic agent is an inhibitorof transforming growth factor-beta (TGF-beta or TGFß). Inhibitors ofTGF-beta proteins being studied which may be used in the presentinvention include NIS793 (Novartis), an anti-TGF-beta antibody beingtested in the clinic for treatment of various cancers, including breast,lung, hepatocellular, colorectal, pancreatic, prostate and renal cancer(NCT02947165). In some embodiments, the inhibitor of TGF-beta proteinsis fresolimumab (GC1008; Sanofi-Genzyme), which is being studied formelanoma (NCT00923169); renal cell carcinoma (NCT00356460); andnon-small cell lung cancer (NCT02581787). Additionally, in someembodiments, the additional therapeutic agent is a TGF-beta trap, suchas described in Connolly et al. (2012) Int'l J. Biological Sciences8:964-978. One therapeutic compound currently in clinical trials fortreatment of solid tumors is M7824 (Merck KgaA—formerly MSB0011459X),which is a bispecific, anti-PD-L1/TGFß trap compound (NCT02699515); and(NCT02517398). M7824 is comprised of a fully human IgG1 antibody againstPD-L1 fused to the extracellular domain of human TGF-beta receptor II,which functions as a TGFß “trap.”

In some embodiments, one or more other therapeutic agent is selectedfrom glembatumumab vedotin-monomethyl auristatin E (MMAE) (Celldex), ananti-glycoprotein NMB (gpNMB) antibody (CR011) linked to the cytotoxicMMAE. gpNMB is a protein overexpressed by 5 multiple tumor typesassociated with cancer cells' ability to metastasize.

In some embodiments, one or more other therapeutic agent is anantiproliferative compound. Such antiproliferative compounds include,but are not limited to: aromatase inhibitors; antiestrogens;topoisomerase I inhibitors; topoisomerase II inhibitors; microtubuleactive compounds; alkylating compounds; histone deacetylase inhibitors;compounds which induce cell differentiation processes; cyclooxygenaseinhibitors; MMP inhibitors; mTOR inhibitors; antineoplasticantimetabolites; platin compounds; compounds targeting/decreasing aprotein or lipid kinase activity and further anti-angiogenic compounds;compounds which target, decrease or inhibit the activity of a protein orlipid phosphatase; gonadorelin agonists; anti-androgens; methionineaminopeptidase inhibitors; matrix metalloproteinase inhibitors;bisphosphonates; biological response modifiers; antiproliferativeantibodies; heparanase inhibitors; inhibitors of Ras oncogenic isoforms;telomerase inhibitors; proteasome inhibitors; compounds used in thetreatment of hematologic malignancies; compounds which target, decreaseor inhibit the activity of Flt-3; Hsp90 inhibitors such as 17-AAG(17-allylaminogeldanamycin, NSC330507), 17-DMAG(17-dimethylaminoethylamino-17-demethoxy-geldanamycin, NSC707545),IPI-504, CNF1010, CNF2024, CNF1010 from Conforma Therapeutics;temozolomide (Temodal®); kinesin spindle protein inhibitors, such asSB715992 or SB743921 from GlaxoSmithKline, or pentamidine/chlorpromazinefrom CombinatoRx; MEK inhibitors such as ARRY142886 from ArrayBioPharma, AZd₆244 from AstraZeneca, PD181461 from Pfizer andleucovorin.

The term “aromatase inhibitor” as used herein relates to a compoundwhich inhibits estrogen production, for instance, the conversion of thesubstrates androstenedione and testosterone to estrone and estradiol,respectively. The term includes, but is not limited to steroids,especially atamestane, exemestane and formestane and, in particular,non-steroids, especially aminoglutethimide, roglethimide,pyridoglutethimide, trilostane, testolactone, ketokonazole, vorozole,fadrozole, anastrozole and letrozole. Exemestane is marketed under thetrade name Aromasin™. Formestane is marketed under the trade nameLentaron™. Fadrozole is marketed under the trade name Afema™.Anastrozole is marketed under the trade name Arimidex™. Letrozole ismarketed under the trade names Femara™ or Femar™. Aminoglutethimide ismarketed under the trade name Orimeten™. A combination of the inventioncomprising a chemotherapeutic agent which is an aromatase inhibitor isparticularly useful for the treatment of hormone receptor positivetumors, such as breast tumors.

The term “antiestrogen” as used herein relates to a compound whichantagonizes the effect of estrogens at the estrogen receptor level. Theterm includes, but is not limited to tamoxifen, fulvestrant, raloxifeneand raloxifene hydrochloride. Tamoxifen is marketed under the trade nameNolvadex™. Raloxifene hydrochloride is marketed under the trade nameEvista™. Fulvestrant can be administered under the trade name Faslodex™.A combination of the invention comprising a chemotherapeutic agent whichis an antiestrogen is particularly useful for the treatment of estrogenreceptor positive tumors, such as breast tumors.

The term “anti-androgen” as used herein relates to any substance whichis capable of inhibiting the biological effects of androgenic hormonesand includes, but is not limited to, bicalutamide (Casodex™). The term“gonadorelin agonist” as used herein includes, but is not limited toabarelix, goserelin and goserelin acetate. Goserelin can be administeredunder the trade name Zoladex™.

The term “topoisomerase I inhibitor” as used herein includes, but is notlimited to topotecan, gimatecan, irinotecan, camptothecian and itsanalogues, 9-nitrocamptothecin and the macromolecular camptothecinconjugate PNU-166148. Irinotecan can be administered, e.g. in the formas it is marketed, e.g. under the trademark Camptosar™. Topotecan ismarketed under the trade name Hycamptin™.

The term “topoisomerase II inhibitor” as used herein includes, but isnot limited to the anthracyclines such as doxorubicin (includingliposomal formulation, such as Caelyx™), daunorubicin, epirubicin,idarubicin and nemorubicin, the anthraquinones mitoxantrone andlosoxantrone, and the podophillotoxines etoposide and teniposide.Etoposide is marketed under the trade name Etopophos™. Teniposide ismarketed under the trade name VM 26-Bristol. Doxorubicin is marketedunder the trade name Acriblastin™ or Adriamycin™. Epirubicin is marketedunder the trade name Farmorubicin™. Idarubicin is marketed. under thetrade name Zavedos™. Mitoxantrone is marketed under the trade nameNovantron.

The term “microtubule active agent” relates to microtubule stabilizing,microtubule destabilizing compounds and microtublin polymerizationinhibitors including, but not limited to: taxanes, such as paclitaxeland docetaxel; vinca alkaloids, such as vinblastine or vinblastinesulfate, vincristine or vincristine sulfate, and vinorelbine;discodermolides; cochicine and epothilones and derivatives thereof.Paclitaxel is marketed under the trade name Taxol™. Docetaxel ismarketed under the trade name Taxotere™. Vinblastine sulfate is marketedunder the trade name Vinblastin R.P™. Vincristine sulfate is marketedunder the trade name Farmistin™.

The term “alkylating agent” as used herein includes, but is not limitedto, cyclophosphamide, ifosfamide, melphalan or nitrosourea (BCNU orGliadel). Cyclophosphamide is marketed under the trade name Cyclostin™.Ifosfamide is marketed under the trade name Holoxan™.

The term “histone deacetylase inhibitors” or “HDAC inhibitors” relatesto compounds which inhibit the histone deacetylase and which possessantiproliferative activity. This includes, but is not limited to,suberoylanilide hydroxamic acid (SAHA).

The term “antineoplastic antimetabolite” includes, but is not limitedto, 5-fluorouracil or 5-FU, capecitabine, gemcitabine, DNA demethylatingcompounds, such as 5-azacytidine and decitabine, methotrexate andedatrexate, and folic acid antagonists such as pemetrexed. Capecitabineis marketed under the trade name Xeloda™. Gemcitabine is marketed underthe trade name Gemzar™.

The term “platin compound” as used herein includes, but is not limitedto, carboplatin, cisplatin, cisplatinum and oxaliplatin. Carboplatin canbe administered, e.g., in the form as it is marketed, e.g. under thetrademark Carboplat™. Oxaliplatin can be administered, e.g., in the formas it is marketed, e.g. under the trademark Eloxatin™.

The term “compounds targeting/decreasing a protein or lipid kinaseactivity; or a protein or lipid phosphatase activity; or furtheranti-angiogenic compounds” as used herein includes, but is not limitedto, protein tyrosine kinase and/or serine and/or threonine kinaseinhibitors or lipid kinase inhibitors, such as: a) compounds targeting,decreasing or inhibiting the activity of the platelet-derived growthfactor-receptors (PDGFR), such as compounds which target, decrease orinhibit the activity of PDGFR, especially compounds which inhibit thePDGF receptor, such as an N-phenyl-2-pyrimidine-amine derivative, suchas imatinib, SU101, SU6668 and GFB-111; b) compounds targeting,decreasing or inhibiting the activity of the fibroblast growthfactor-receptors (FGFR); c) compounds targeting, decreasing orinhibiting the activity of the insulin-like growth factor receptor I(IGF-IR), such as compounds which target, decrease or inhibit theactivity of IGF-IR, especially compounds which inhibit the kinaseactivity of IGF-I receptor, or antibodies that target the extracellulardomain of IGF-I receptor or its growth factors; d) compounds targeting,decreasing or inhibiting the activity of the Trk receptor tyrosinekinase family, or ephrin B4 inhibitors; e) compounds targeting,decreasing or inhibiting the activity of the Axl receptor tyrosinekinase family; f) compounds targeting, decreasing or inhibiting theactivity of the Ret receptor tyrosine kinase; g) compounds targeting,decreasing or inhibiting the activity of the Kit/SCFR receptor tyrosinekinase, such as imatinib; h) compounds targeting, decreasing orinhibiting the activity of the C-kit receptor tyrosine kinases, whichare part of the PDGFR family, such as compounds which target, decreaseor inhibit the activity of the c-Kit receptor tyrosine kinase family,especially compounds which inhibit the c-Kit receptor, such as imatinib;i) compounds targeting, decreasing or inhibiting the activity of membersof the c-Abl family, their gene-fusion products (e.g. BCR-Abl kinase)and mutants, such as compounds which target decrease or inhibit theactivity of c-Abl family members and their gene fusion products, such asan N-phenyl-2-pyrimidine-amine derivative, such as imatinib or nilotinib(AMN107); PD180970; AG957; NSC 680410; PD173955 from ParkeDavis; ordasatinib (BMS-354825); j) compounds targeting, decreasing or inhibitingthe activity of members of the protein kinase C (PKC) and Raf family ofserine/threonine kinases, members of the MEK, SRC, JAK/pan-JAK, FAK,PDK1, PKB/Akt, Ras/MAPK, PI3K, SYK, TYK2, BTK and TEC family, and/ormembers of the cyclin-dependent kinase family (CDK) includingstaurosporine derivatives, such as midostaurin; examples of furthercompounds include UCN-01, safingol, BAY 43-9006, Bryostatin 1,Perifosine; Ilmofosine; RO 318220 and RO 320432; GO 6976; Isis 3521;LY333531/LY379196; isochinoline compounds; FTIs; PD184352 or QAN697 (aP13K inhibitor) or AT7519 (CDK inhibitor); k) compounds targeting,decreasing or inhibiting the activity of protein-tyrosine kinaseinhibitors, such as compounds which target, decrease or inhibit theactivity of protein-tyrosine kinase inhibitors include imatinib mesylate(Gleevec™) or tyrphostin such as Tyrphostin A23/RG-50810; AG 99;Tyrphostin AG 213; Tyrphostin AG 1748; Tyrphostin AG 490; TyrphostinB44; Tyrphostin B44 (+) enantiomer; Tyrphostin AG 555; AG 494;Tyrphostin AG 556, AG957 and adaphostin(4-{[(2,5-dihydroxyphenyl)methyl]amino}-benzoic acid adamantyl ester;NSC 680410, adaphostin); I) compounds targeting, decreasing orinhibiting the activity of the epidermal growth factor family ofreceptor tyrosine kinases (EGFR₁ ErbB2, ErbB3, ErbB4 as homo- orheterodimers) and their mutants, such as compounds which target,decrease or inhibit the activity of the epidermal growth factor receptorfamily are especially compounds, proteins or antibodies which inhibitmembers of the EGF receptor tyrosine kinase family, such as EGFreceptor, ErbB2, ErbB3 and ErbB4 or bind to EGF or EGF related ligands,CP 358774, ZD 1839, ZM 105180; trastuzumab (Herceptin™), cetuximab(Erbitux™), Iressa, Tarceva, OSI-774, CI-1033, EKB-569, GW-2016, E1.1,E2.4, E2.5, E6.2, E6.4, E2.11, E6.3 or E7.6.3, and7H-pyrrolo-[2,3-d]pyrimidine derivatives; m) compounds targeting,decreasing or inhibiting the activity of the c-Met receptor, such ascompounds which target, decrease or inhibit the activity of c-Met,especially compounds which inhibit the kinase activity of c-Metreceptor, or antibodies that target the extracellular domain of c-Met orbind to HGF, n) compounds targeting, decreasing or inhibiting the kinaseactivity of one or more JAK family members (JAK1/JAK2/JAK3/TYK2 and/orpan-JAK), including but not limited to PRT-062070, SB-1578, baricitinib,pacritinib, momelotinib, VX-509, AZD-1480, TG-101348, tofacitinib, andruxolitinib; o) compounds targeting, decreasing or inhibiting the kinaseactivity of PI3 kinase (PI3K) including but not limited to ATU-027,SF-1126, DS-7423, PBI-05204, GSK-2126458, ZSTK-474, buparlisib,pictrelisib, PF-4691502, BYL-719, dactolisib, XL-147, XL-765, andidelalisib; and; and p) compounds targeting, decreasing or inhibitingthe signaling effects of hedgehog protein (Hh) or smoothened receptor(SMO) pathways, including but not limited to cyclopamine, vismodegib,itraconazole, erismodegib, and IPI-926 (saridegib).

The term “PI3K inhibitor” as used herein includes, but is not limited tocompounds having inhibitory activity against one or more enzymes in thephosphatidylinositol-3-kinase family, including, but not limited toPI3Kα, PI3Kγ, PI3Kδ, PI3Kβ, PI3K-C2α, PI3K-C2β, PI3K-C2γ, Vps34, p110-α,p110-β, p110-γ, p110-δ, p85-α, p85-β, p55-γ, p150, p101, and p87.Examples of PI3K inhibitors useful in this invention include but are notlimited to ATU-027, SF-1126, DS-7423, PBI-05204, GSK-2126458, ZSTK-474,buparlisib, pictrelisib, PF-4691502, BYL-719, dactolisib, XL-147,XL-765, and idelalisib.

The term “Bcl-2 inhibitor” as used herein includes, but is not limitedto compounds having inhibitory activity against B-cell lymphoma 2protein (Bcl-2), including but not limited to ABT-199, ABT-731, ABT-737,apogossypol, Ascenta's pan-Bcl-2 inhibitors, curcumin (and analogsthereof), dual Bcl-2/Bcl-xL inhibitors (InfinityPharmaceuticals/Novartis Pharmaceuticals), Genasense (G3139), HA14-1(and analogs thereof; see WO 2008/118802), navitoclax (and analogsthereof, see U.S. Pat. No. 7,390,799), NH-1 (Shenayng PharmaceuticalUniversity), obatoclax (and analogs thereof, see WO 2004/106328), S-001(Gloria Pharmaceuticals), TW series compounds (Univ. of Michigan), andvenetoclax. In some embodiments the Bcl-2 inhibitor is a small moleculetherapeutic. In some embodiments the Bcl-2 inhibitor is apeptidomimetic.

The term “BTK inhibitor” as used herein includes, but is not limited tocompounds having inhibitory activity against Bruton's Tyrosine Kinase(BTK), including, but not limited to AVL-292 and ibrutinib.

The term “SYK inhibitor” as used herein includes, but is not limited tocompounds having inhibitory activity against spleen tyrosine kinase(SYK), including but not limited to PRT-062070, R-343, R-333, Excellair,PRT-062607, and fostamatinib.

Further examples of BTK inhibitory compounds, and conditions treatableby such compounds in combination with compounds of this invention can befound in WO 2008/039218 and WO 2011/090760, the entirety of which areincorporated herein by reference.

Further examples of SYK inhibitory compounds, and conditions treatableby such compounds in combination with compounds of this invention can befound in WO 2003/063794, WO 2005/007623, and WO 2006/078846, theentirety of which are incorporated herein by reference.

Further examples of PI3K inhibitory compounds, and conditions treatableby such compounds in combination with compounds of this invention can befound in WO 2004/019973, WO 2004/089925, WO 2007/016176, U.S. Pat. No.8,138,347, WO 2002/088112, WO 2007/084786, WO 2007/129161, WO2006/122806, WO 2005/113554, and WO 2007/044729 the entirety of whichare incorporated herein by reference.

Further examples of JAK inhibitory compounds, and conditions treatableby such compounds in combination with compounds of this invention can befound in WO 2009/114512, WO 2008/109943, WO 2007/053452, WO 2000/142246,and WO 2007/070514, the entirety of which are incorporated herein byreference.

Further anti-angiogenic compounds include compounds having anothermechanism for their activity, e.g. unrelated to protein or lipid kinaseinhibition e.g. thalidomide (Thalomid™) and TNP-470.

Examples of proteasome inhibitors useful for use in combination withcompounds of the invention include, but are not limited to bortezomib,disulfiram, epigallocatechin-3-gallate (EGCG), salinosporamide A,carfilzomib, ONX-0912, CEP-18770, and MLN9708.

Compounds which target, decrease or inhibit the activity of a protein orlipid phosphatase are e.g. inhibitors of phosphatase 1, phosphatase 2A,or CDC25, such as okadaic acid or a derivative thereof.

Compounds which induce cell differentiation processes include, but arenot limited to, retinoic acid, α- γ- or δ-tocopherol or α- γ- orδ-tocotrienol.

The term cyclooxygenase inhibitor as used herein includes, but is notlimited to, Cox-2 inhibitors, 5-alkyl substituted2-arylaminophenylacetic acid and derivatives, such as celecoxib(Celebrex™), rofecoxib (Vioxx™), etoricoxib, valdecoxib or a5-alkyl-2-arylaminophenylacetic acid, such as5-methyl-2-(2′-chloro-6′-fluoroanilino)phenyl acetic acid andlumiracoxib.

The term “bisphosphonates” as used herein includes, but is not limitedto, etridonic, clodronic, tiludronic, pamidronic, alendronic,ibandronic, risedronic and zoledronic acid. Etridonic acid is marketedunder the trade name Didronel™. Clodronic acid is marketed under thetrade name Bonefos™. Tiludronic acid is marketed under the trade nameSkelid™. Pamidronic acid is marketed under the trade name Aredia™.Alendronic acid is marketed under the trade name Fosamax™. Ibandronicacid is marketed under the trade name Bondranat™. Risedronic acid ismarketed under the trade name Actonel™. Zoledronic acid is marketedunder the trade name Zometa™. The term “mTOR inhibitors” relates tocompounds which inhibit the mammalian target of rapamycin (mTOR) andwhich possess antiproliferative activity such as sirolimus (Rapamune®),everolimus (Certican™), CCI-779 and ABT578.

The term “heparanase inhibitor” as used herein refers to compounds whichtarget, decrease or inhibit heparin sulfate degradation. The termincludes, but is not limited to, PI-88. The term “biological responsemodifier” as used herein refers to a lymphokine or interferons.

The term “inhibitor of Ras oncogenic isoforms”, such as H-Ras, K-Ras, orN-Ras, as used herein refers to compounds which target, decrease orinhibit the oncogenic activity of Ras; for example, a “farnesyltransferase inhibitor” such as L-744832, DK8G557 or R115777(Zarnestra™). The term “telomerase inhibitor” as used herein refers tocompounds which target, decrease or inhibit the activity of telomerase.Compounds which target, decrease or inhibit the activity of telomeraseare especially compounds which inhibit the telomerase receptor, such astelomestatin.

The term “methionine aminopeptidase inhibitor” as used herein refers tocompounds which target, decrease or inhibit the activity of methionineaminopeptidase. Compounds which target, decrease or inhibit the activityof methionine aminopeptidase include, but are not limited to, bengamideor a derivative thereof.

The term “proteasome inhibitor” as used herein refers to compounds whichtarget, decrease or inhibit the activity of the proteasome. Compoundswhich target, decrease or inhibit the activity of the proteasomeinclude, but are not limited to, Bortezomib (Velcade™) and MLN 341.

The term “matrix metalloproteinase inhibitor” or (“MMP” inhibitor) asused herein includes, but is not limited to, collagen peptidomimetic andnonpeptidomimetic inhibitors, tetracycline derivatives, e.g. hydroxamatepeptidomimetic inhibitor batimastat and its orally bioavailable analoguemarimastat (BB-2516), prinomastat (AG3340), metastat (NSC 683551)BMS-279251, BAY 12-9566, TAA211, MMI270B or AAJ996.

The term “compounds used in the treatment of hematologic malignancies”as used herein includes, but is not limited to, FMS-like tyrosine kinaseinhibitors, which are compounds targeting, decreasing or inhibiting theactivity of FMS-like tyrosine kinase receptors (Flt-3R); interferon,1-β-D-arabinofuransylcytosine (ara-c) and bisulfan; and ALK inhibitors,which are compounds which target, decrease or inhibit anaplasticlymphoma kinase.

Compounds which target, decrease or inhibit the activity of FMS-liketyrosine kinase receptors (Flt-3R) are especially compounds, proteins orantibodies which inhibit members of the Flt-3R receptor kinase family,such as PKC412, midostaurin, a staurosporine derivative, SU11248 andMLN518.

The term “HSP90 inhibitors” as used herein includes, but is not limitedto, compounds targeting, decreasing or inhibiting the intrinsic ATPaseactivity of HSP90; degrading, targeting, decreasing or inhibiting theHSP90 client proteins via the ubiquitin proteosome pathway. Compoundstargeting, decreasing or inhibiting the intrinsic ATPase activity ofHSP90 are especially compounds, proteins or antibodies which inhibit theATPase activity of HSP90, such as 17-allylamino,17-demethoxygeldanamycin (17AAG), a geldanamycin derivative; othergeldanamycin related compounds; radicicol and HDAC inhibitors.

The term “antiproliferative antibodies” as used herein includes, but isnot limited to, trastuzumab (Herceptin™), Trastuzumab-DM1, erbitux,bevacizumab (Avastin™), rituximab (Rituxan®), PRO64553 (anti-CD40) and2C4 Antibody. By antibodies is meant intact monoclonal antibodies,polyclonal antibodies, multispecific antibodies formed from at least 2intact antibodies, and antibody fragments so long as they exhibit thedesired biological activity.

For the treatment of acute myeloid leukemia (AML), compounds of thecurrent invention can be used in combination with standard leukemiatherapies, especially in combination with therapies used for thetreatment of AML. In particular, compounds of the current invention canbe administered in combination with, for example, farnesyl transferaseinhibitors and/or other drugs useful for the treatment of AML, such asDaunorubicin, Adriamycin, Ara-C, VP-16, Teniposide, Mitoxantrone,Idarubicin, Carboplatinum and PKC412.

Other anti-leukemic compounds include, for example, Ara-C, a pyrimidineanalog, which is the 2′-alpha-hydroxy ribose (arabinoside) derivative ofdeoxycytidine. Also included is the purine analog of hypoxanthine,6-mercaptopurine (6-MP) and fludarabine phosphate. Compounds whichtarget, decrease or inhibit activity of histone deacetylase (HDAC)inhibitors such as sodium butyrate and suberoylanilide hydroxamic acid(SAHA) inhibit the activity of the enzymes known as histonedeacetylases. Specific HDAC inhibitors include MS275, SAHA, FK228(formerly FR901228), Trichostatin A and compounds disclosed in U.S. Pat.No. 6,552,065 including, but not limited to,N-hydroxy-3-[4-[[[2-(2-methyl-1H-indol-3-yl)-ethyl]-amino]methyl]phenyl]-2E-2-propenamide,or a pharmaceutically acceptable salt thereof andN-hydroxy-3-[4-[(2-hydroxyethyl){2-(1H-indol-3-yl)ethyl]-amino]methyl]phenyl]-2E-2-propenamide,or a pharmaceutically acceptable salt thereof, especially the lactatesalt. Somatostatin receptor antagonists as used herein refer tocompounds which target, treat or inhibit the somatostatin receptor suchas octreotide, and SOM230. Tumor cell damaging approaches refer toapproaches such as ionizing radiation. The term “ionizing radiation”referred to above and hereinafter means ionizing radiation that occursas either electromagnetic rays (such as X-rays and gamma rays) orparticles (such as alpha and beta particles). Ionizing radiation isprovided in, but not limited to, radiation therapy and is known in theart. See Hellman, Principles of Radiation Therapy, Cancer, in Principlesand Practice of Oncology, Devita et al., Eds., 4^(th) Edition, Vol. 1,pp. 248-275 (1993).

Also included are EDG binders and ribonucleotide reductase inhibitors.The term “EDG binders” as used herein refers to a class ofimmunosuppressants that modulates lymphocyte recirculation, such asFTY720. The term “ribonucleotide reductase inhibitors” refers topyrimidine or purine nucleoside analogs including, but not limited to,fludarabine and/or cytosine arabinoside (ara-C), 6-thioguanine,5-fluorouracil, cladribine, 6-mercaptopurine (especially in combinationwith ara-C against ALL) and/or pentostatin. Ribonucleotide reductaseinhibitors are especially hydroxyurea or2-hydroxy-1H-isoindole-1,3-dione derivatives.

Also included are in particular those compounds, proteins or monoclonalantibodies of VEGF such as:1-(4-chloroanilino)-4-(4-pyridylmethyl)phthalazine or a pharmaceuticallyacceptable salt thereof,1-(4-chloroanilino)-4-(4-pyridylmethyl)phthalazine succinate;Angiostatin™; Endostatin™; anthranilic acid amides; ZD4190; Zd₆474;SU5416; SU6668; bevacizumab; or anti-VEGF antibodies or anti-VEGFreceptor antibodies, such as rhuMAb and RHUFab, VEGF aptamer such asMacugon; FLT-4 inhibitors, FLT-3 inhibitors, VEGFR-2 IgGI antibody,Angiozyme (RPI 4610) and Bevacizumab (Avastin™).

Photodynamic therapy as used herein refers to therapy which uses certainchemicals known as photosensitizing compounds to treat or preventcancers. Examples of photodynamic therapy include treatment withcompounds, such as Visudyne™ and porfimer sodium.

Angiostatic steroids as used herein refers to compounds which block orinhibit angiogenesis, such as, e.g., anecortave, triamcinolone,hydrocortisone, 11-α-epihydrocotisol, cortexolone,17α-hydroxyprogesterone, corticosterone, desoxycorticosterone,testosterone, estrone and dexamethasone.

Implants containing corticosteroids refers to compounds, such asfluocinolone and dexamethasone.

Other chemotherapeutic compounds include, but are not limited to: plantalkaloids, hormonal compounds and antagonists; biological responsemodifiers, preferably lymphokines or interferons; antisenseoligonucleotides or oligonucleotide derivatives; shRNA or siRNA; ormiscellaneous compounds or compounds with other or unknown mechanism ofaction.

The structure of the active compounds identified by code numbers,generic or trade names may be taken from the actual edition of thestandard compendium “The Merck Index” or from databases, e.g. PatentsInternational (e.g. IMS World Publications).

Exemplary Immuno-Oncology Agents

In some embodiments, one or more other therapeutic agent is animmuno-oncology agent. As used herein, the term “an immuno-oncologyagent” refers to an agent which is effective to enhance, stimulate,and/or up-regulate immune responses in a subject. In some embodiments,the administration of an immuno-oncology agent with a compound of theinvention has a synergic effect in treating a cancer.

An immuno-oncology agent can be, for example, a small molecule drug, anantibody, or a biologic or small molecule. Examples of biologicimmuno-oncology agents include, but are not limited to, cancer vaccines,antibodies, and cytokines. In some embodiments, an antibody is amonoclonal antibody. In some embodiments, a monoclonal antibody ishumanized or human.

In some embodiments, an immuno-oncology agent is (i) an agonist of astimulatory (including a co-stimulatory) receptor or (ii) an antagonistof an inhibitory (including a co-inhibitory) signal on T cells, both ofwhich result in amplifying antigen-specific T cell responses.

Certain of the stimulatory and inhibitory molecules are members of theimmunoglobulin super family (IgSF). One important family ofmembrane-bound ligands that bind to co-stimulatory or co-inhibitoryreceptors is the B7 family, which includes B7-1, B7-2, B7-H1 (PD-L1),B7-DC (PD-L2), B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5 (VISTA), and B7-H6.Another family of membrane bound ligands that bind to co-stimulatory orco-inhibitory receptors is the TNF family of molecules that bind tocognate TNF receptor family members, which includes CD40 and CD40L,OX-40, OX-40L, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137 (4-1BB),TRAIL/Apo2-L, TRAILR1/DR4, TRAILR2/DR5, TRAILR3, TRAILR4, OPG, RANK,RANKL, TWEAKR/Fn14, TWEAK, BAFFR, EDAR, XEDAR, TACI, APRIL, BCMA, LTβR,LIGHT, DcR3, HVEM, VEGI/TL1A, TRAMP/DR3, EDAR, EDA1, XEDAR, EDA2, TNFR1,Lymphotoxin α/TNFβ, TNFR2, TNFα, LTβR, Lymphotoxin α1β2, FAS, FASL,RELT, DR6, TROY and NGFR.

In some embodiments, an immuno-oncology agent is a cytokine thatinhibits T cell activation (e.g., IL-6, IL-10, TGF-β, VEGF, and otherimmunosuppressive cytokines) or a cytokine that stimulates T cellactivation, for stimulating an immune response.

In some embodiments, a combination of a compound of the invention and animmuno-oncology agent can stimulate T cell responses. In someembodiments, an immuno-oncology agent is: (i) an antagonist of a proteinthat inhibits T cell activation (e.g., immune checkpoint inhibitors)such as CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, TIM-3, Galectin 9, CEACAM-1,BTLA, CD69, Galectin-1, TIGIT, CD113, GPR56, VISTA, 2B4, CD48, GARP,PD1H, LAIR1, TIM-1, and TIM-4; or (ii) an agonist of a protein thatstimulates T cell activation such as B7-1, B7-2, CD28, 4-1BB (CD137),4-1BBL, ICOS, ICOS-L, OX40, OX40L, GITR, GITRL, CD70, CD27, CD40, DR3and CD28H.

In some embodiments, an immuno-oncology agent is an antagonist ofinhibitory receptors on NK cells or an agonist of activating receptorson NK cells. In some embodiments, an immuno-oncology agent is anantagonist of KIR, such as lirilumab.

In some embodiments, an immuno-oncology agent is an agent that inhibitsor depletes macrophages or monocytes, including but not limited toCSF-1R antagonists such as CSF-1R antagonist antibodies including RG7155(WO 2011/70024, WO 2011/107553, WO 2011/131407, WO 2013/87699, WO2013/119716, WO 2013/132044) or FPA-008 (WO 2011/140249; WO 2013/169264;WO 2014/036357).

In some embodiments, an immuno-oncology agent is selected from agonisticagents that ligate positive costimulatory receptors, blocking agentsthat attenuate signaling through inhibitory receptors, antagonists, andone or more agents that increase systemically the frequency ofanti-tumor T cells, agents that overcome distinct immune suppressivepathways within the tumor microenvironment (e.g., block inhibitoryreceptor engagement (e.g., PD-L1/PD-1 interactions), deplete or inhibitTregs (e.g., using an anti-CD25 monoclonal antibody (e.g., daclizumab)or by ex vivo anti-CD25 bead depletion), inhibit metabolic enzymes suchas IDO, or reverse/prevent T cell energy or exhaustion) and agents thattrigger innate immune activation and/or inflammation at tumor sites.

In some embodiments, an immuno-oncology agent is a CTLA-4 antagonist. Insome embodiments, a CTLA-4 antagonist is an antagonistic CTLA-4antibody. In some embodiments, an antagonistic CTLA-4 antibody is YERVOY(ipilimumab) or tremelimumab.

In some embodiments, an immuno-oncology agent is a PD-1 antagonist. Insome embodiments, a PD-1 antagonist is administered by infusion. In someembodiments, an immuno-oncology agent is an antibody or anantigen-binding portion thereof that binds specifically to a ProgrammedDeath-1 (PD-1) receptor and inhibits PD-1 activity. In some embodiments,a PD-1 antagonist is an antagonistic PD-1 antibody. In some embodiments,an antagonistic PD-1 antibody is OPDIVO (nivolumab), KEYTRUDA(pembrolizumab), or MEDI-0680 (AMP-514; WO 2012/145493). In someembodiments, an immuno-oncology agent may be pidilizumab (CT-011). Insome embodiments, an immuno-oncology agent is a recombinant proteincomposed of the extracellular domain of PD-L2 (B7-DC) fused to the Fcportion of IgG1, called AMP-224.

In some embodiments, an immuno-oncology agent is a PD-L1 antagonist. Insome embodiments, a PD-L1 antagonist is an antagonistic PD-L1 antibody.In some embodiments, a PD-L1 antibody is MPDL3280A (RG7446; WO2010/077634), durvalumab (MEDI4736), BMS-936559 (WO 2007/005874), andMSB0010718C (WO 2013/79174).

In some embodiments, an immuno-oncology agent is a LAG-3 antagonist. Insome embodiments, a LAG-3 antagonist is an antagonistic LAG-3 antibody.In some embodiments, a LAG3 antibody is BMS-986016 (WO 2010/19570, WO2014/08218), or IMP-731 or IMP-321 (WO 2008/132601, WO 2009/44273).

In some embodiments, an immuno-oncology agent is a CD137 (4-1BB)agonist. In some embodiments, a CD137 (4-1BB) agonist is an agonisticCD137 antibody. In some embodiments, a CD137 antibody is urelumab orPF-05082566 (WO 2012/32433).

In some embodiments, an immuno-oncology agent is a GITR agonist. In someembodiments, a GITR agonist is an agonistic GITR antibody. In someembodiments, a GITR antibody is BMS-986153, BMS-986156, TRX-518 (WO2006/105021, WO 2009/009116), or MK-4166 (WO 2011/028683).

In some embodiments, an immuno-oncology agent is an indoleamine(2,3)-dioxygenase (IDO) antagonist. In some embodiments, an IDOantagonist is selected from: epacadostat (INCB024360, Incyte); indoximod(NLG-8189, NewLink Genetics Corporation); capmanitib (INC280, Novartis);GDC-0919 (Genentech/Roche); PF-06840003 (Pfizer); BMS:F001287(Bristol-Myers Squibb); Phy906/KD108 (Phytoceutica); an enzyme thatbreaks down kynurenine (Kynase, Kyn Therapeutics); and NLG-919 (WO2009/73620, WO 2009/1156652, WO 2011/56652, WO 2012/142237).

In some embodiments, an immuno-oncology agent is an OX40 agonist. Insome embodiments, an OX40 agonist is an agonistic OX40 antibody. In someembodiments, an OX40 antibody is MEDI-6383 or MEDI-6469.

In some embodiments, an immuno-oncology agent is an OX40L antagonist. Insome embodiments, an OX40L antagonist is an antagonistic OX40 antibody.In some embodiments, an OX40L antagonist is RG-7888 (WO 2006/029879).

In some embodiments, an immuno-oncology agent is a CD40 agonist. In someembodiments, a CD40 agonist is an agonistic CD40 antibody. In someembodiments, an immuno-oncology agent is a CD40 antagonist. In someembodiments, a CD40 antagonist is an antagonistic CD40 antibody. In someembodiments, a CD40 antibody is lucatumumab or dacetuzumab.

In some embodiments, an immuno-oncology agent is a CD27 agonist. In someembodiments, a CD27 agonist is an agonistic CD27 antibody. In someembodiments, a CD27 antibody is varlilumab.

In some embodiments, an immuno-oncology agent is MGA271 (to B7H3) (WO2011/109400).

In some embodiments, an immuno-oncology agent is abagovomab,adecatumumab, afutuzumab, alemtuzumab, anatumomab mafenatox, apolizumab,atezolimab, avelumab, blinatumomab, BMS-936559, catumaxomab, durvalumab,epacadostat, epratuzumab, indoximod, inotuzumab ozogamicin, intelumumab,ipilimumab, isatuximab, lambrolizumab, MED14736, MPDL3280A, nivolumab,obinutuzumab, ocaratuzumab, ofatumumab, olatatumab, pembrolizumab,pidilizumab, rituximab, ticilimumab, samalizumab, or tremelimumab.

In some embodiments, an immuno-oncology agent is an immunostimulatoryagent. For example, antibodies blocking the PD-1 and PD-L1 inhibitoryaxis can unleash activated tumor-reactive T cells and have been shown inclinical trials to induce durable anti-tumor responses in increasingnumbers of tumor histologies, including some tumor types thatconventionally have not been considered immunotherapy sensitive. See,e.g., Okazaki, T. et al. (2013) Nat. Immunol. 14, 1212-1218; Zou et al.(2016) Sci. Transl. Med. 8. The anti-PD-1 antibody nivolumab (Opdivo®,Bristol-Myers Squibb, also known as ONO-4538, MDX1106 and BMS-936558),has shown potential to improve the overall survival in patients withrenal clear cell carcinoma (RCC) who had experienced disease progressionduring or after prior anti-angiogenic therapy.

In some embodiments, the immunomodulatory therapeutic specificallyinduces apoptosis of tumor cells. Approved immunomodulatory therapeuticswhich may be used in the present invention include: pomalidomide(Pomalyst®, Celgene); lenalidomide (Revlimid®, Celgene); ingenolmebutate (Picato®, LEO Pharma).

In some embodiments, an immuno-oncology agent is a cancer vaccine. Insome embodiments, the cancer vaccine is selected from: sipuleucel-T(Provenge®, Dendreon/Valeant Pharmaceuticals), which has been approvedfor treatment of asymptomatic, or minimally symptomatic metastaticcastrate-resistant (hormone-refractory) prostate cancer; and talimogenelaherparepvec (Imlygic®, BioVex/Amgen, previously known as T-VEC), agenetically modified oncolytic viral therapy approved for treatment ofunresectable cutaneous, subcutaneous and nodal lesions in melanoma. Insome embodiments, an immuno-oncology agent is selected from an oncolyticviral therapy such as pexastimogene devacirepvec (PexaVec/JX-594,SillaJen/formerly Jennerex Biotherapeutics), a thymidine kinase- (TK-)deficient vaccinia virus engineered to express GM-CSF, forhepatocellular carcinoma (NCT02562755) and melanoma (NCT00429312);pelareorep (Reolysin®, Oncolytics Biotech), a variant of respiratoryenteric orphan virus (reovirus) which does not replicate in cells thatare not RAS-activated, in numerous cancers, including colorectal cancer(NCT01622543); prostate cancer (NCT01619813); head and neck squamouscell cancer (NCT01166542); pancreatic adenocarcinoma (NCT00998322); andnon-small cell lung cancer (NSCLC) (NCT 00861627); enadenotucirev(NG-348, PsiOxus, formerly known as ColoAd1), an adenovirus engineeredto express a full length CD80 and an antibody fragment specific for theT-cell receptor CD3 protein, in ovarian cancer (NCT02028117); metastaticor advanced epithelial tumors such as in colorectal cancer, bladdercancer, head and neck squamous cell carcinoma and salivary gland cancer(NCT02636036); ONCOS-102 (Targovax/formerly Oncos), an adenovirusengineered to express GM-CSF, in melanoma (NCT03003676); and peritonealdisease, colorectal cancer or ovarian cancer (NCT02963831); GL-ONC1(GLV-1h68/GLV-1h153, Genelux GmbH), vaccinia viruses engineered toexpress beta-galactosidase (beta-gal)/beta-glucoronidase orbeta-gal/human sodium iodide symporter (hNIS), respectively, werestudied in peritoneal carcinomatosis (NCT01443260); fallopian tubecancer, ovarian cancer (NCT 02759588); or CG0070 (Cold Genesys), anadenovirus engineered to express GM-CSF, in bladder cancer(NCT02365818).

In some embodiments, an immuno-oncology agent is selected from: JX-929(SillaJen/formerly Jennerex Biotherapeutics), a TK- and vaccinia growthfactor-deficient vaccinia virus engineered to express cytosinedeaminase, which is able to convert the prodrug 5-fluorocytosine to thecytotoxic drug 5-fluorouracil; TG01 and TG02 (Targovax/formerly Oncos),peptide-based immunotherapy agents targeted for difficult-to-treat RASmutations; and TILT-123 (TILT Biotherapeutics), an engineered adenovirusdesignated: Ad5/3-E2F-delta24-hTNFα-IRES-hIL20; and VSV-GP(ViraTherapeutics) a vesicular stomatitis virus (VSV) engineered toexpress the glycoprotein (GP) of lymphocytic choriomeningitis virus(LCMV), which can be further engineered to express antigens designed toraise an antigen-specific CD8⁺ T cell response.

In some embodiments, an immuno-oncology agent is a T-cell engineered toexpress a chimeric antigen receptor, or CAR. The T-cells engineered toexpress such chimeric antigen receptor are referred to as CAR-T cells.

CARs have been constructed that consist of binding domains, which may bederived from natural ligands, single chain variable fragments (scFv)derived from monoclonal antibodies specific for cell-surface antigens,fused to endodomains that are the functional end of the T-cell receptor(TCR), such as the CD3-zeta signaling domain from TCRs, which is capableof generating an activation signal in T lymphocytes. Upon antigenbinding, such CARs link to endogenous signaling pathways in the effectorcell and generate activating signals similar to those initiated by theTCR complex.

For example, in some embodiments the CAR-T cell is one of thosedescribed in U.S. Pat. No. 8,906,682 (hereby incorporated by referencein its entirety), which discloses CAR-T cells engineered to comprise anextracellular domain having an antigen binding domain (such as a domainthat binds to CD19), fused to an intracellular signaling domain of the Tcell antigen receptor complex zeta chain (such as CD3 zeta). Whenexpressed in the T cell, the CAR is able to redirect antigen recognitionbased on the antigen binding specificity. In the case of CD19, theantigen is expressed on malignant B cells. Over 200 clinical trials arecurrently in progress employing CAR-T in a wide range of indications.[https://clinicaltrials.gov/ct2/results?term=chimeric+antigen+receptors&pg=1].

In some embodiments, an immunostimulatory agent is an activator ofretinoic acid receptor-related orphan receptor γ (RORγt). RORγt is atranscription factor with key roles in the differentiation andmaintenance of Type 17 effector subsets of CD4+ (Th17) and CD8+ (Tc17) Tcells, as well as the differentiation of IL-17 expressing innate immunecell subpopulations such as NK cells. In some embodiments, an activatorof RORγt is LYC-55716 (Lycera), which is currently being evaluated inclinical trials for the treatment of solid tumors (NCT02929862).

In some embodiments, an immunostimulatory agent is an agonist oractivator of a toll-like receptor (TLR). Suitable activators of TLRsinclude an agonist or activator of TLR9 such as SD-101 (Dynavax). SD-101is an immunostimulatory CpG which is being studied for B-cell,follicular and other lymphomas (NCT02254772). Agonists or activators ofTLR8 which may be used in the present invention include motolimod(VTX-2337, VentiRx Pharmaceuticals) which is being studied for squamouscell cancer of the head and neck (NCT02124850) and ovarian cancer(NCT02431559).

Other immuno-oncology agents that may be used in the present inventioninclude: urelumab (BMS-663513, Bristol-Myers Squibb), an anti-CD137monoclonal antibody; varlilumab (CDX-1127, Celldex Therapeutics), ananti-CD27 monoclonal antibody; BMS-986178 (Bristol-Myers Squibb), ananti-OX40 monoclonal antibody; lirilumab (IPH2102/BMS-986015, InnatePharma, Bristol-Myers Squibb), an anti-KIR monoclonal antibody;monalizumab (IPH2201, Innate Pharma, AstraZeneca) an anti-NKG2Amonoclonal antibody; andecaliximab (GS-5745, Gilead Sciences), ananti-MMP9 antibody; and MK-4166 (Merck & Co.), an anti-GITR monoclonalantibody.

In some embodiments, an immunostimulatory agent is selected fromelotuzumab, mifamurtide, an agonist or activator of a toll-likereceptor, and an activator of RORγt.

In some embodiments, an immunostimulatory therapeutic is recombinanthuman interleukin 15 (rhIL-15). rhIL-15 has been tested in the clinic asa therapy for melanoma and renal cell carcinoma (NCT01021059 andNCT01369888) and leukemias (NCT02689453). In some embodiments, animmunostimulatory agent is recombinant human interleukin 12 (rhIL-12).In some embodiments, an IL-15 based immunotherapeutic is heterodimericIL-15 (hetIL-15, Novartis/Admune), a fusion complex composed of asynthetic form of endogenous IL-15 complexed to the soluble IL-15binding protein IL-15 receptor alpha chain (IL15:sIL-15RA), which hasbeen tested in Phase 1 clinical trials for melanoma, renal cellcarcinoma, non-small cell lung cancer and head and neck squamous cellcarcinoma (NCT02452268). In some embodiments, a recombinant humaninterleukin 12 (rhIL-12) is NM-IL-12 (Neumedicines, Inc.), NCT02544724,or NCT02542124.

In some embodiments, an immuno-oncology agent is selected from thosedescribed in Jerry L. Adams et al., “Big opportunities for smallmolecules in immuno-oncology,” Cancer Therapy 2015, Vol. 14, pages603-622, the content of which is incorporated herein by reference in itsentirety. In some embodiment, an immuno-oncology agent is selected fromthe examples described in Table 1 of Jerry L. Adams et al. In someembodiments, an immuno-oncology agent is a small molecule targeting animmuno-oncology target selected from those listed in Table 2 of Jerry L.Adams et al. In some embodiments, an immuno-oncology agent is a smallmolecule agent selected from those listed in Table 2 of Jerry L. Adamset al.

In some embodiments, an immuno-oncology agent is selected from the smallmolecule immuno-oncology agents described in Peter L. Toogood, “Smallmolecule immuno-oncology therapeutic agents,” Bioorganic & MedicinalChemistry Letters 2018, Vol. 28, pages 319-329, the content of which isincorporated herein by reference in its entirety. In some embodiments,an immuno-oncology agent is an agent targeting the pathways as describedin Peter L. Toogood.

In some embodiments, an immuno-oncology agent is selected from thosedescribed in Sandra L. Ross et al., “Bispecific T cell engager (BiTE®)antibody constructs can mediate bystander tumor cell killing”, PLoS ONE12(8): e0183390, the content of which is incorporated herein byreference in its entirety. In some embodiments, an immuno-oncology agentis a bispecific T cell engager (BiTE®) antibody construct. In someembodiments, a bispecific T cell engager (BiTE®) antibody construct is aCD19/CD3 bispecific antibody construct. In some embodiments, abispecific T cell engager (BiTE®) antibody construct is an EGFR/CD3bispecific antibody construct. In some embodiments, a bispecific T cellengager (BiTE®) antibody construct activates T cells. In someembodiments, a bispecific T cell engager (BiTE®) antibody constructactivates T cells, which release cytokines inducing upregulation ofintercellular adhesion molecule 1 (ICAM-1) and FAS on bystander cells.In some embodiments, a bispecific T cell engager (BiTE®) antibodyconstruct activates T cells which result in induced bystander celllysis. In some embodiments, the bystander cells are in solid tumors. Insome embodiments, the bystander cells being lysed are in proximity tothe BiTE®-activated T cells. In some embodiment, the bystander cellscomprises tumor-associated antigen (TAA) negative cancer cells. In someembodiment, the bystander cells comprise EGFR-negative cancer cells. Insome embodiments, an immuno-oncology agent is an antibody which blocksthe PD-L1/PD1 axis and/or CTLA4. In some embodiments, an immuno-oncologyagent is an ex vivo expanded tumor-infiltrating T cell. In someembodiments, an immuno-oncology agent is a bispecific antibody constructor chimeric antigen receptors (CARs) that directly connect T cells withtumor-associated surface antigens (TAAs).

Exemplary Immune Checkpoint Inhibitors

In some embodiments, an immuno-oncology agent is an immune checkpointinhibitor as described herein.

The term “checkpoint inhibitor” as used herein relates to agents usefulin preventing cancer cells from avoiding the immune system of thepatient. One of the major mechanisms of anti-tumor immunity subversionis known as “T-cell exhaustion,” which results from chronic exposure toantigens that has led to up-regulation of inhibitory receptors. Theseinhibitory receptors serve as immune checkpoints in order to preventuncontrolled immune reactions.

PD-1 and co-inhibitory receptors such as cytotoxic T-lymphocyte antigen4 (CTLA-4, B and T Lymphocyte Attenuator (BTLA; CD272), T cellImmunoglobulin and Mucin domain-3 (Tim-3), Lymphocyte Activation Gene-3(Lag-3; CD223), and others are often referred to as checkpointregulators. They act as molecular “gatekeepers” that allow extracellularinformation to dictate whether cell cycle progression and otherintracellular signaling processes should proceed.

In some embodiments, an immune checkpoint inhibitor is an antibody toPD-1. PD-1 binds to the programmed cell death 1 receptor (PD-1) toprevent the receptor from binding to the inhibitory ligand PDL-1, thusoverriding the ability of tumors to suppress the host anti-tumor immuneresponse.

In one aspect, the checkpoint inhibitor is a biologic therapeutic or asmall molecule. In another aspect, the checkpoint inhibitor is amonoclonal antibody, a humanized antibody, a fully human antibody, afusion protein or a combination thereof. In a further aspect, thecheckpoint inhibitor inhibits a checkpoint protein selected from CTLA-4,PDLI, PDL2, PDI, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR,2B4, CD160, CGEN-15049, CHK 1, CHK2, A2aR, B-7 family ligands or acombination thereof. In an additional aspect, the checkpoint inhibitorinteracts with a ligand of a checkpoint protein selected from CTLA-4,PDLI, PDL2, PDI, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR,2B4, CD160, CGEN-15049, CHK 1, CHK2, A2aR, B-7 family ligands or acombination thereof. In an aspect, the checkpoint inhibitor is animmunostimulatory agent, a T cell growth factor, an interleukin, anantibody, a vaccine or a combination thereof. In a further aspect, theinterleukin is IL-7 or IL-15. In a specific aspect, the interleukin isglycosylated IL-7. In an additional aspect, the vaccine is a dendriticcell (DC) vaccine.

Checkpoint inhibitors include any agent that blocks or inhibits in astatistically significant manner, the inhibitory pathways of the immunesystem. Such inhibitors may include small molecule inhibitors or mayinclude antibodies, or antigen binding fragments thereof, that bind toand block or inhibit immune checkpoint receptors or antibodies that bindto and block or inhibit immune checkpoint receptor ligands. Illustrativecheckpoint molecules that may be targeted for blocking or inhibitioninclude, but are not limited to, CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4,BTLA, HVEM, GAL9, LAG3, TIM3, VISTA, KIR, 2B4 (belongs to the CD2 familyof molecules and is expressed on all NK, γδ, and memory CD8⁺ (αβ) Tcells), CD160 (also referred to as BY55), CGEN-15049, CHK 1 and CHK2kinases, A2aR, and various B-7 family ligands. B7 family ligandsinclude, but are not limited to, B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3,B7-H4, B7-H5, B7-H6 and B7-H7. Checkpoint inhibitors include antibodies,or antigen binding fragments thereof, other binding proteins, biologictherapeutics, or small molecules, that bind to and block or inhibit theactivity of one or more of CTLA-4, PDL1, PDL2, PD1, BTLA, HVEM, TIM3,GAL9, LAG3, VISTA, KIR, 2B4, CD 160 and CGEN-15049. Illustrative immunecheckpoint inhibitors include Tremelimumab (CTLA-4 blocking antibody),anti-OX40, PD-LI monoclonal Antibody (Anti-B7-HI; MEDI4736), MK-3475(PD-1 blocker), Nivolumab (anti-PDI antibody), CT-011 (anti-PDIantibody), BY55 monoclonal antibody, AMP224 (anti-PDLI antibody),BMS-936559 (anti-PDLI antibody), MPLDL3280A (anti-PDLI antibody),MSB0010718C (anti-PDLI antibody), and ipilimumab (anti-CTLA-4 checkpointinhibitor). Checkpoint protein ligands include, but are not limited toPD-LI, PD-L2, B7-H3, B7-H4, CD28, CD86 and TIM-3.

In certain embodiments, the immune checkpoint inhibitor is selected froma PD-1 antagonist, a PD-L1 antagonist, and a CTLA-4 antagonist. In someembodiments, the checkpoint inhibitor is selected from the groupconsisting of nivolumab (Opdivo®), ipilimumab (Yervoy®), andpembrolizumab (Keytruda®). In some embodiments, the checkpoint inhibitoris selected from: nivolumab (anti-PD-1 antibody, Opdivo®, Bristol-MyersSquibb); pembrolizumab (anti-PD-1 antibody, Keytruda®, Merck);ipilimumab (anti-CTLA-4 antibody, Yervoy®, Bristol-Myers Squibb);durvalumab (anti-PD-L1 antibody, Imfinzi®, AstraZeneca); andatezolizumab (anti-PD-L1 antibody, Tecentriq®, Genentech).

In some embodiments, the checkpoint inhibitor is selected from the groupconsisting of lambrolizumab (MK-3475), nivolumab (BMS-936558),pidilizumab (CT-011), AMP-224, MDX-1105, MEDI4736, MPDL3280A,BMS-936559, ipilimumab, lirlumab, IPH2101, pembrolizumab (Keytruda®),and tremelimumab.

In some embodiments, an immune checkpoint inhibitor is: REGN2810(Regeneron), an anti-PD-1 antibody tested in patients with basal cellcarcinoma (NCT03132636); NSCLC (NCT03088540); cutaneous squamous cellcarcinoma (NCT02760498); lymphoma (NCT02651662); and melanoma(NCT03002376); pidilizumab (CureTech), also known as CT-011, an antibodythat binds to PD-1, in clinical trials for diffuse large B-cell lymphomaand multiple myeloma; avelumab (Bavencio®, Pfizer/Merck KGaA), alsoknown as MSB0010718C), a fully human IgG1 anti-PD-L1 antibody, inclinical trials for non-small cell lung cancer, Merkel cell carcinoma,mesothelioma, solid tumors, renal cancer, ovarian cancer, bladdercancer, head and neck cancer, and gastric cancer; or PDR001 (Novartis),an inhibitory antibody that binds to PD-1, in clinical trials fornon-small cell lung cancer, melanoma, triple negative breast cancer andadvanced or metastatic solid tumors. Tremelimumab (CP-675,206;Astrazeneca) is a fully human monoclonal antibody against CTLA-4 thathas been studied in clinical trials for a number of indications,including: mesothelioma, colorectal cancer, kidney cancer, breastcancer, lung cancer and non-small cell lung cancer, pancreatic ductaladenocarcinoma, pancreatic cancer, germ cell cancer, squamous cellcancer of the head and neck, hepatocellular carcinoma, prostate cancer,endometrial cancer, metastatic cancer in the liver, liver cancer, largeB-cell lymphoma, ovarian cancer, cervical cancer, metastatic anaplasticthyroid cancer, urothelial cancer, fallopian tube cancer, multiplemyeloma, bladder cancer, soft tissue sarcoma, and melanoma. AGEN-1884(Agenus) is an anti-CTLA4 antibody that is being studied in Phase 1clinical trials for advanced solid tumors (NCT02694822).

In some embodiments, a checkpoint inhibitor is an inhibitor of T-cellimmunoglobulin mucin containing protein-3 (TIM-3). TIM-3 inhibitors thatmay be used in the present invention include TSR-022, LY3321367 andMBG453. TSR-022 (Tesaro) is an anti-TIM-3 antibody which is beingstudied in solid tumors (NCT02817633). LY3321367 (Eli Lilly) is ananti-TIM-3 antibody which is being studied in solid tumors(NCT03099109). MBG453 (Novartis) is an anti-TIM-3 antibody which isbeing studied in advanced malignancies (NCT02608268).

In some embodiments, a checkpoint inhibitor is an inhibitor of T cellimmunoreceptor with Ig and ITIM domains, or TIGIT, an immune receptor oncertain T cells and NK cells. TIGIT inhibitors that may be used in thepresent invention include BMS-986207 (Bristol-Myers Squibb), ananti-TIGIT monoclonal antibody (NCT02913313); OMP-313M32 (Oncomed); andanti-TIGIT monoclonal antibody (NCT03119428).

In some embodiments, a checkpoint inhibitor is an inhibitor ofLymphocyte Activation Gene-3 (LAG-3). LAG-3 inhibitors that may be usedin the present invention include BMS-986016 and REGN3767 and IMP321.BMS-986016 (Bristol-Myers Squibb), an anti-LAG-3 antibody, is beingstudied in glioblastoma and gliosarcoma (NCT02658981). REGN3767(Regeneron), is also an anti-LAG-3 antibody, and is being studied inmalignancies (NCT03005782). IMP321 (Immutep S.A.) is an LAG-3-Ig fusionprotein, being studied in: melanoma (NCT02676869); adenocarcinoma(NCT02614833); and metastatic breast cancer (NCT00349934).

Checkpoint inhibitors that may be used in the present invention includeOX40 agonists. OX40 agonists that are being studied in clinical trialsinclude: PF-04518600/PF-8600 (Pfizer), an agonistic anti-OX40 antibody,in metastatic kidney cancer (NCT03092856) and advanced cancers andneoplasms (NCT02554812; NCT05082566); GSK3174998 (Merck), an agonisticanti-OX40 antibody, in Phase 1 cancer trials (NCT02528357); MEDI0562(Medimmune/AstraZeneca), an agonistic anti-OX40 antibody, in advancedsolid tumors (NCT02318394 and NCT02705482); MEDI6469, an agonisticanti-OX40 antibody (Medimmune/AstraZeneca), in patients with colorectalcancer (NCT02559024), breast cancer (NCT01862900), head and neck cancer(NCT02274155) and metastatic prostate cancer (NCT01303705); andBMS-986178 (Bristol-Myers Squibb) an agonistic anti-OX40 antibody, inadvanced cancers (NCT02737475).

Checkpoint inhibitors that may be used in the present invention includeCD137 (also called 4-1BB) agonists. CD137 agonists that are beingstudied in clinical trials include: utomilumab (PF-05082566, Pfizer) anagonistic anti-CD137 antibody, in diffuse large B-cell lymphoma(NCT02951156) and in advanced cancers and neoplasms (NCT02554812 andNCT05082566); urelumab (BMS-663513, Bristol-Myers Squibb), an agonisticanti-CD137 antibody, in melanoma and skin cancer (NCT02652455) andglioblastoma and gliosarcoma (NCT02658981).

Checkpoint inhibitors that may be used in the present invention includeCD27 agonists. CD27 agonists that are being studied in clinical trialsinclude: varlilumab (CDX-1127, Celldex Therapeutics) an agonisticanti-CD27 antibody, in squamous cell head and neck cancer, ovariancarcinoma, colorectal cancer, renal cell cancer, and glioblastoma(NCT02335918); lymphomas (NCT01460134); and glioma and astrocytoma(NCT02924038).

Checkpoint inhibitors that may be used in the present invention includeglucocorticoid-induced tumor necrosis factor receptor (GITR) agonists.GITR agonists that are being studied in clinical trials include: TRX518(Leap Therapeutics), an agonistic anti-GITR antibody, in malignantmelanoma and other malignant solid tumors (NCT01239134 and NCT02628574);GWN323 (Novartis), an agonistic anti-GITR antibody, in solid tumors andlymphoma (NCT02740270); INCAGN01876 (Incyte/Agenus), an agonisticanti-GITR antibody, in advanced cancers (NCT02697591 and NCT03126110);MK-4166 (Merck), an agonistic anti-GITR antibody, in solid tumors(NCT02132754) and MEDI1873 (Medimmune/AstraZeneca), an agonistichexameric GITR-ligand molecule with a human IgG1 Fc domain, in advancedsolid tumors (NCT02583165).

Checkpoint inhibitors that may be used in the present invention includeinducible T-cell co-stimulator (ICOS, also known as CD278) agonists.ICOS agonists that are being studied in clinical trials include:MEDI-570 (Medimmune), an agonistic anti-ICOS antibody, in lymphomas(NCT02520791); GSK3359609 (Merck), an agonistic anti-ICOS antibody, inPhase 1 (NCT02723955); and JTX-2011 (Jounce Therapeutics), an agonisticanti-ICOS antibody, in Phase 1 (NCT02904226).

Checkpoint inhibitors that may be used in the present invention includekiller IgG-like receptor (KIR) inhibitors. KIR inhibitors that are beingstudied in clinical trials include: lirilumab (IPH2102/BMS-986015,Innate Pharma/Bristol-Myers Squibb), an anti-KIR antibody, in leukemias(NCT01687387, NCT02399917, NCT02481297, NCT02599649), multiple myeloma(NCT02252263), and lymphoma (NCT01592370); IPH2101 (1-7F9, InnatePharma) in myeloma (NCT01222286 and NCT01217203); and IPH4102 (InnatePharma), an anti-KIR antibody that binds to three domains of the longcytoplasmic tail (KIR3DL2), in lymphoma (NCT02593045).

Checkpoint inhibitors that may be used in the present invention includeCD47 inhibitors of interaction between CD47 and signal regulatoryprotein alpha (SIRPa). CD47/SIRPa inhibitors that are being studied inclinical trials include: ALX-148 (Alexo Therapeutics), an antagonisticvariant of (SIRPa) that binds to CD47 and prevents CD47/SIRPa-mediatedsignaling, in phase 1 (NCT03013218); TTI-621 (SIRPa-Fc, TrilliumTherapeutics), a soluble recombinant fusion protein created by linkingthe N-terminal CD47-binding domain of SIRPa with the Fc domain of humanIgG1, acts by binding human CD47, and preventing it from delivering its“do not eat” signal to macrophages, is in clinical trials in Phase 1(NCT02890368 and NCT02663518); CC-90002 (Celgene), an anti-CD47antibody, in leukemias (NCT02641002); and Hu5F9-G4 (Forty Seven, Inc.),in colorectal neoplasms and solid tumors (NCT02953782), acute myeloidleukemia (NCT02678338) and lymphoma (NCT02953509).

Checkpoint inhibitors that may be used in the present invention includeCD73 inhibitors. CD73 inhibitors that are being studied in clinicaltrials include: MEDI19447 (Medimmune), an anti-CD73 antibody, in solidtumors (NCT02503774); and BMS-986179 (Bristol-Myers Squibb), ananti-CD73 antibody, in solid tumors (NCT02754141).

Checkpoint inhibitors that may be used in the present invention includeagonists of stimulator of interferon genes protein (STING, also knownastransmembrane protein 173, or TMEM173). Agonists of STING that arebeing studied in clinical trials include: MK-1454 (Merck), an agonisticsynthetic cyclic dinucleotide, in lymphoma (NCT03010176); and ADU-S100(MIW815, Aduro Biotech/Novartis), an agonistic synthetic cyclicdinucleotide, in Phase 1 (NCT02675439 and NCT03172936).

Checkpoint inhibitors that may be used in the present invention includeCSF1R inhibitors. CSF1R inhibitors that are being studied in clinicaltrials include: pexidartinib (PLX3397, Plexxikon), a CSF1R smallmolecule inhibitor, in colorectal cancer, pancreatic cancer, metastaticand advanced cancers (NCT02777710) and melanoma, non-small cell lungcancer, squamous cell head and neck cancer, gastrointestinal stromaltumor (GIST) and ovarian cancer (NCT02452424); and IMC-CS4 (LY3022855,Lilly), an anti-CSF-1R antibody, in pancreatic cancer (NCT03153410),melanoma (NCT03101254), and solid tumors (NCT02718911); and BLZ945(4-[2((1R,2R)-2-hydroxycyclohexylamino)-benzothiazol-6-yloxyl]-pyridine-2-carboxylicacid methylamide, Novartis), an orally available inhibitor of CSF1R, inadvanced solid tumors (NCT02829723).

Checkpoint inhibitors that may be used in the present invention includeNKG2A receptor inhibitors. NKG2A receptor inhibitors that are beingstudied in clinical trials include monalizumab (IPH2201, Innate Pharma),an anti-NKG2A antibody, in head and neck neoplasms (NCT02643550) andchronic lymphocytic leukemia (NCT02557516).

In some embodiments, the immune checkpoint inhibitor is selected fromnivolumab, pembrolizumab, ipilimumab, avelumab, durvalumab,atezolizumab, or pidilizumab.

Therapeutic Uses

The bicyclic peptides of the invention have specific utility as Nectin-4binding agents.

Nectin-4 is a surface molecule that belongs to the nectin family ofproteins, which comprises 4 members. Nectins are cell adhesion moleculesthat play a key role in various biological processes such as polarity,proliferation, differentiation and migration, for epithelial,endothelial, immune and neuronal cells, during development and adultlife. They are involved in several pathological processes in humans.They are the main receptors for poliovirus, herpes simplex virus andmeasles virus. Mutations in the genes encoding Nectin-1 (PVRL1) orNectin-4 (PVRL4) cause ectodermal dysplasia syndromes associated withother abnormalities. Nectin-4 is expressed during foetal development. Inadult tissues its expression is more restricted than that of othermembers of the family. Nectin-4 is a tumour-associated antigen in 50%,49% and 86% of breast, ovarian and lung carcinomas, respectively, mostlyon tumours of bad prognosis. Its expression is not detected in thecorresponding normal tissues. In breast tumours, Nectin-4 is expressedmainly in triple-negative and ERBB2+ carcinomas. In the serum ofpatients with these cancers, the detection of soluble forms of Nectin-4is associated with a poor prognosis. Levels of serum Nectin-4 increaseduring metastatic progression and decrease after treatment. Theseresults suggest that Nectin-4 could be a reliable target for thetreatment of cancer. Accordingly, several anti-Nectin-4 antibodies havebeen described in the prior art. In particular, Enfortumab Vedotin(ASG-22ME) is an antibody-drug conjugate (ADC) targeting Nectin-4 and iscurrently clinically investigated for the treatment of patientssuffering from solid tumours.

Polypeptide ligands selected according to the method of the presentinvention may be employed in in vivo therapeutic and prophylacticapplications, in vitro and in vivo diagnostic applications, in vitroassay and reagent applications, and the like. Ligands having selectedlevels of specificity are useful in applications which involve testingin non-human animals, where cross-reactivity is desirable, or indiagnostic applications, where cross-reactivity with homologues orparalogues needs to be carefully controlled. In some applications, suchas vaccine applications, the ability to elicit an immune response topredetermined ranges of antigens can be exploited to tailor a vaccine tospecific diseases and pathogens.

Substantially pure peptide ligands of at least 90 to 95% homogeneity arepreferred for administration to a mammal, and 98 to 99% or morehomogeneity is most preferred for pharmaceutical uses, especially whenthe mammal is a human. Once purified, partially or to homogeneity asdesired, the selected polypeptides may be used diagnostically ortherapeutically (including extracorporeally) or in developing andperforming assay procedures, immunofluorescent stainings and the like(Lefkovite and Pernis, (1979 and 1981) Immunological Methods, Volumes Iand II, Academic Press, NY).

According to a further aspect of the invention, there is provided apeptide ligand or a drug conjugate as defined herein, for use inpreventing, suppressing or treating a disease or disorder mediated byNectin-4.

According to a further aspect of the invention, there is provided amethod of preventing, suppressing or treating a disease or disordermediated by Nectin-4, which comprises administering to a patient in needthereof an effector group and drug conjugate of the peptide ligand asdefined herein.

In one embodiment, the Nectin-4 is mammalian Nectin-4. In a furtherembodiment, the mammalian Nectin-4 is human Nectin-4.

In one embodiment, the disease or disorder mediated by Nectin-4 isselected from viral infections, ectodermal dysplasia syndromes and otherabnormalities, breast, ovarian and lung carcinomas, metastaticprogression, and solid tumours.

In a further embodiment, the disease or disorder mediated by Nectin-4 isselected from cancer.

Examples of cancers (and their benign counterparts) which may be treated(or inhibited) include, but are not limited to tumours of epithelialorigin (adenomas and carcinomas of various types includingadenocarcinomas, squamous carcinomas, transitional cell carcinomas andother carcinomas) such as carcinomas of the bladder and urinary tract,breast, gastrointestinal tract (including the esophagus, stomach(gastric), small intestine, colon, rectum and anus), liver(hepatocellular carcinoma), gall bladder and biliary system, exocrinepancreas, kidney, lung (for example adenocarcinomas, small cell lungcarcinomas, non-small cell lung carcinomas, bronchioalveolar carcinomasand mesotheliomas), head and neck (for example cancers of the tongue,buccal cavity, larynx, pharynx, nasopharynx, tonsil, salivary glands,nasal cavity and paranasal sinuses), ovary, fallopian tubes, peritoneum,vagina, vulva, penis, cervix, myometrium, endometrium, thyroid (forexample thyroid follicular carcinoma), adrenal, prostate, skin andadnexae (for example melanoma, basal cell carcinoma, squamous cellcarcinoma, keratoacanthoma, dysplastic naevus); haematologicalmalignancies (i.e. leukemias, lymphomas) and premalignant haematologicaldisorders and disorders of borderline malignancy includinghaematological malignancies and related conditions of lymphoid lineage(for example acute lymphocytic leukemia [ALL], chronic lymphocyticleukemia [CLL], B-cell lymphomas such as diffuse large B-cell lymphoma[DLBCL], follicular lymphoma, Burkitt's lymphoma, mantle cell lymphoma,T-cell lymphomas and leukaemias, natural killer [NK] cell lymphomas,Hodgkin's lymphomas, hairy cell leukaemia, monoclonal gammopathy ofuncertain significance, plasmacytoma, multiple myeloma, andpost-transplant lymphoproliferative disorders), and haematologicalmalignancies and related conditions of myeloid lineage (for exampleacute myelogenousleukemia [AML], chronic myelogenousleukemia [CML],chronic myelomonocyticleukemia [CMML], hypereosinophilic syndrome,myeloproliferative disorders such as polycythaemia vera, essentialthrombocythaemia and primary myelofibrosis, myeloproliferative syndrome,myelodysplastic syndrome, and promyelocyticleukemia); tumours ofmesenchymal origin, for example sarcomas of soft tissue, bone orcartilage such as osteosarcomas, fibrosarcomas, chondrosarcomas,rhabdomyosarcomas, leiomyosarcomas, liposarcomas, angiosarcomas,Kaposi's sarcoma, Ewing's sarcoma, synovial sarcomas, epithelioidsarcomas, gastrointestinal stromal tumours, benign and malignanthistiocytomas, and dermatofibrosarcomaprotuberans; tumours of thecentral or peripheral nervous system (for example astrocytomas, gliomasand glioblastomas, meningiomas, ependymomas, pineal tumours andschwannomas); endocrine tumours (for example pituitary tumours, adrenaltumours, islet cell tumours, parathyroid tumours, carcinoid tumours andmedullary carcinoma of the thyroid); ocular and adnexal tumours (forexample retinoblastoma); germ cell and trophoblastic tumours (forexample teratomas, seminomas, dysgerminomas, hydatidiform moles andchoriocarcinomas); and paediatric and embryonal tumours (for examplemedulloblastoma, neuroblastoma, Wilms tumour, and primitiveneuroectodermal tumours); or syndromes, congenital or otherwise, whichleave the patient susceptible to malignancy (for example XerodermaPigmentosum).

In a further embodiment, the cancer is selected from a hematopoieticmalignancy such as selected from: non-Hodgkin's lymphoma (NHL),Burkitt's lymphoma (BL), multiple myeloma (MM), B chronic lymphocyticleukemia (B-CLL), B and T acute lymphocytic leukemia (ALL), T celllymphoma (TCL), acute myeloid leukemia (AML), hairy cell leukemia (HCL),Hodgkin's Lymphoma (HL), and chronic myeloid leukemia (CML).

In a yet further embodiment, the cancer is selected from lung cancer(e.g. non-small cell lung cancer), bladder cancer, pancreatic cancer andbreast cancer. Data is presented herein in Examples 1 to 5 whichdemonstrates that selected bicyclic drug conjugates of the inventionexhibited anti-tumour activity in these cancer models.

References herein to the term “prevention” involves administration ofthe protective composition prior to the induction of the disease.“Suppression” refers to administration of the composition after aninductive event, but prior to the clinical appearance of the disease.“Treatment” involves administration of the protective composition afterdisease symptoms become manifest.

Animal model systems which can be used to screen the effectiveness ofthe peptide ligands in protecting against or treating the disease areavailable. The use of animal model systems is facilitated by the presentinvention, which allows the development of polypeptide ligands which cancross react with human and animal targets, to allow the use of animalmodels.

Furthermore, data is presented herein which demonstrates an associationbetween copy number variation (CNV) and gene expression for Nectin-4from multiple tumor types. Thus, according to a further aspect of theinvention, there is provided a method of preventing, suppressing ortreating cancer, which comprises administering to a patient in needthereof an effector group and drug conjugate of the peptide ligand asdefined herein, wherein said patient is identified as having anincreased copy number variation (CNV) of Nectin-4.

In one embodiment, the cancer is selected from those identified hereinas having increased CNV of Nectin-4. In a further embodiment, the canceris selected from those identified herein as having increased CNV ofNectin-4, namely: breast, uterine, bladder, lung adenocarcinoma, lungsquamous, cervical, head and neck, pancreatic, thyroid, colorectal,thymoma, sarcoma, renal clear cell carcinoma (RCC), prostate andstomach.

The invention is further described below with reference to the followingexamples.

EXAMPLES Abbreviations

-   1,2,4-TriAz 3-(1,2,4-Triazol-1-yl)-alanine-   1Nal 1-Naphthylalanine-   2FuAla 2-Furylalanine-   2MePhe 2-Methyl-Phenylalanine-   2Nal 2-Naphthylalanine-   2Pal 2-Pyridylalanine-   3,3-DPA 3,3-Diphenylalanine-   3MePhe 3-Methyl-Phenylalanine-   3Pal 3-Pyridylalanine-   4,4-BPA 4,4-Biphenylalanine-   4,4-DFP 4,4-Difluoroproline-   4MePhe 4-Methyl-Phenylalanine-   4Pal 4-Pyridylalanine-   4ThiAz Beta-(4-Thiazolyl)-Alanine-   5FTrp 5-Fluoro-L-tryptophan-   Agb 2-Amino-4-guanidinobutyric acid-   Aib Aminoisobutyric acid-   AzaTrp Azatryptophan-   Aze Azetidine-   C5A Cyclopentyl glycine-   Cha 3-Cyclohexyl-alanine-   Cpa Cyclopropylalanine-   Cya Cysteic acid-   DOPA 3,4-Dihydroxy-phenylalanine-   HArg HomoArginine-   HGln HomoGlutamine-   Hleu HomeLeucine-   Hphe HomoPhenylalanine-   Hse(me) Homoserine(Me)-   HSer HomoSerine-   HyP Hydroxyproline-   Lys(Ac) Lysine(Acetyl)-   Met(O2) Methionine sulfone-   Nle Norleucine-   Oic Octahydroindolecarboxylic acid-   Oxa Oxazolidine-4-carboxylic acid-   pCoPhe para-Carboxy-Phenylalanine-   PheOPhe 4-Phenoxy-phenylalanine-   Phg Phenylglycine-   Pip Pipecolic acid-   Pro(4NH) 4-Amino-Proline-   tBuAla t-Butyl-Alanine-   TetraZ Tetrazole Alanine-   Thi Thienyl-alanine-   THP(O) Tetrahydropyran-4-propanoic acid-   THP(SO2) Dioxo-4-tetrahydrothiopyranylacetic acid-   Trp(Me) Methyl Trptophan    Materials and Methods    Peptide Synthesis

Peptide synthesis was based on Fmoc chemistry, using a Symphony peptidesynthesiser manufactured by Peptide Instruments and a Syro IIsynthesiser by MultiSynTech. Standard Fmoc-amino acids were employed(Sigma, Merck), with appropriate side chain protecting groups: whereapplicable standard coupling conditions were used in each case, followedby deprotection using standard methodology.

Alternatively, peptides were purified using HPLC and following isolationthey were modified with 1,3,5-Triacryloylhexahydro-1,3,5-triazine (TATA,Sigma). For this, linear peptide was diluted with 50:50 MeCN:H₂O up to˜35 mL, ˜500 μL of 100 mM TATA in acetonitrile was added, and thereaction was initiated with 5 mL of 1 M NH₄HCO₃ in H₂O. The reaction wasallowed to proceed for ˜30-60 min at RT, and lyophilised once thereaction had completed (judged by MALDI). Once completed, 1 ml of 1ML-cysteine hydrochloride monohydrate (Sigma) in H₂O was added to thereaction for ˜60 min at RT to quench any excess TATA. Followinglyophilisation, the modified peptide was purified as above, whilereplacing the Luna C8 with a Gemini C18 column (Phenomenex), andchanging the acid to 0.1% trifluoroacetic acid. Pure fractionscontaining the correct TATA-modified material were pooled, lyophilisedand kept at −20° C. for storage.

All amino acids, unless noted otherwise, were used in theL-configurations.

In some cases peptides are converted to activated disulfides prior tocoupling with the free thiol group of a toxin using the followingmethod; a solution of 4-methyl(succinimidyl 4-(2-pyridylthio)pentanoate)(100 mM) in dry DMSO (1.25 mol equiv) was added to a solution of peptide(20 mM) in dry DMSO (1 mol equiv). The reaction was well mixed and DIPEA(20 mol equiv) was added. The reaction was monitored by LC/MS untilcomplete.

Preparation of Bicyclic Peptide Drug Conjugates

Preparation of BCY8549

Separation Condition: A phase: 0.075% TFA in H₂O, B phase: MeCN

Separation method: 18-48-55 min, RT=53.5 min

Separation column: Luna 200*25 mm 10 μm, C18, 110 A and Gemin150*30 mm,C18, 5 μm, 110 A, connection, 50° C.

Dissolve method: DMF

Separation purity: 95%

BCY8234 was synthesized by solid phase synthesis.

Preparation of Compound 2

The peptide was synthesized using standard Fmoc chemistry.

-   1) Add DCM to the vessel containing CTC Resin (5 mmol, 4.3 g, 1.17    mmol/g) and Fmoc-Cit —OH (2.0 g, 5 mmol, 1.0 eq) with N₂ bubbling.-   2) Add DIEA (4.0 eq) dropwise and mix for 2 hours.-   3) Add MeOH (5 mL) and mix for 30 min.-   4) Drain and wash with DMF for 5 times.-   5) Add 20% piperidine/DMF and react on 30 min.-   6) Drain and wash with DMF for 5 times.-   7) Add Fmoc-amino acid solution and mix 30 seconds, then add    activation buffer, N₂ bubbling for about 1 hour.-   8) Repeat above step 4 to 7 for the coupling of following amino    acids.

Note:

# Materials Coupling reagents 1 Fmoc-Cit-OH (1.0 eq) DIEA (4.0 eq) 21-ethoxycarbonylcyclobutanecarboxylic HATU (2.85 eq) and acid (3.0 eq)DIEA (6.0 eq)

20% piperidine in DMF was used for Fmoc deprotection for 30 min. Thecoupling reaction was monitored by ninhydrin test, and the resin waswashed with DMF for 5 times.

Peptide Cleavage and Purification:

-   1) Add cleavage buffer (20% TFIP/80% DCM) to the flask containing    the side chain protected peptide at room temperature and stir for 1    hour twice.-   2) Filter and collect the filtrate.-   3) Concentrate to remove the solvent.-   4) The crude peptide was lyophilized to give the final product (1.4    g, 85.0% yield).    Preparation of Compound 3

To a solution of compound 2 (1.65 g, 5.01 mmol, 1.0 eq) in DCM (30 mL)and MeOH (15 mL) was added EEDQ (2.48 g, 10.02 mmol, 2.0 eq) and(4-aminophenyl)methanol (740.37 mg, 6.01 mmol, 1.2 eq). The mixture wasstirred at 15° C. for 16 hr. LC-MS showed compound 2 was consumedcompletely and one main peak with desired m/z was detected. TLCindicated compound 2 was consumed completely and many new spots formed.The reaction mixture was concentrated under reduced pressure to removesolvent to give a residue. The residue was purified by flash silica gelchromatography (ISCO®; 80 SepaFlash® Silica Flash Column, Eluent of 0˜15DCM/MeOH gradient @ 60 mL/min). Compound 3 (1.3 g, 2.99 mmol, 59.72%yield) was obtained as a yellow solid.

Preparation of Compound 4

To a solution of compound 3 (1.3 g, 2.99 mmol, 1.0 eq) in DMF (10 mL)was added DIEA (2.32 g, 17.95 mmol, 3.13 mL, 6.0 eq) andbis(4-nitrophenyl) carbonate (3.64 g, 11.97 mmol, 4.0 eq). The mixturewas stirred at 15° C. for 1 hr. LC-MS showed compound 3 was consumedcompletely and one main peak with desired m/z was detected. The residuewas purified by prep-HPLC (neutral condition). Compound 4 (1.0 g, 1.67mmol, 55.74% yield) was obtained as a yellow solid.

Preparation of Compound 5

To a solution of compound 5 (250.53 mg, 417.84 μmol, 1.5 eq) in DMF (5mL) was added HOBt (56.46 mg, 417.84 μmol, 1.5 eq) and DIEA (108.01 mg,835.68 μmol, 145.56 μL, 3.0 eq), MMAE (0.200 g, 278.56 μmol, 1.0 eq).The mixture was stirred at 35° C. for 12 hr. LC-MS showed MMAE wasconsumed completely and one main peak with desired m/z was detected. Thereaction was directly purified by prep-HPLC (neutral condition).Compound 5 (0.180 g, 152.74 μmol, 54.83% yield) was obtained as a yellowsolid.

Preparation of Compound 6

To a solution of compound 5 (0.170 g, 144.26 μmol, 1.0 eq) in THF (5 mL)and H₂O (5 mL) was added LiOH.H₂O (12.11 mg, 288.51 μmol, 2.0 eq). Themixture was stirred at 15° C. for 1 hr. LC-MS showed compound 5 wasconsumed completely and one main peak with desired m/z was detected.Adjusted PH=7 used by AcOH and THF was removed under reduced pressure togive a residue. The residue was purified by prep-HPLC (neutralcondition). Compound 6 (0.185 g, crude) was obtained as a yellow solid.

Preparation of BCY8549

To a solution of compound 6 (0.100 g, 86.93 μmol, 1.0 eq) in DMA (4 mL)was added HOSu (10.00 mg, 86.93 μmol, 1.0 eq) and EDCI (16.66 mg, 86.93μmol, 1.0 eq). After the NHS ester was formed, β-Ala-BCY8234 (525.98 mg,173.85 μmol, 2.0 eq) and DIEA (33.70 mg, 260.78 μmol, 45.42 μL, 3.0 eq).The mixture was stirred at 15° C. for 4 hr. LC-MS showed compound 6 wasconsumed completely and one main peak with desired m/z was detected. Thereaction was directly purified by prep-HPLC (TFA condition). CompoundBCY8549 (0.0528 g, 12.15 μmol, 13.98% yield, 95.70% purity) was obtainedas a white solid. Retenton time=11.48 min. Mass found=1386.4 (M/3+H)

Preparation of BCY8245

Separation Condition: A phase: 0.075% TFA in H₂O, B phase: MeCN

Separation method: 18-48-55 min, RT=53.5 min

Separation column: Luna 200*25 mm 10 um, C18, 110 A and Gemin150*30 mm,C18, 5 um, 110 A, connection, 50° C.

Dissolve method: DMF

Separation purity: 95%

The BCY8234 was synthesized by solid phase synthesis.

Reaction Scheme of BCY8245 is Shown Below:

Preparation of Compound 3

The compound 3 was synthesized by solid phase method.

Preparation of Compound 4

To a solution of compound 3 (1.3 g, 3.23 mmol, 1.0 eq) in DCM (10 mL)and MeOH (5 mL) was added EEDQ (1.60 g, 6.46 mmol, 2.0 eq) and(4-aminophenyl)methanol (517.16 mg, 4.20 mmol, 1.3 eq). The mixture wasstirred at 20° C. for 16 hr. LC-MS showed compound 3 was consumedcompletely and one main peak with desired m/z was detected. The solventwas removed under reduced pressure. The residue was purified by flashsilica gel chromatography (ISCO®; 40 g SepaFlash® Silica Flash Column,Eluent of 0˜15% DCM/MeOH gradient @ 40 mL/min). Compound 4 (0.950 g,1.87 mmol, 57.94% yield) was obtained as a yellow solid.

Preparation of Compound 5

To a solution of compound 4 (0.950 g, 1.87 mmol, 1.0 eq) in DMF (5 mL)was added DIEA (1.21 g, 9.36 mmol, 1.63 mL, 5.0 eq) andbis(4-nitrophenyl) carbonate (2.28 g, 7.49 mmol, 4.0 eq). The mixturewas stirred at 20° C. for 1 hr. LC-MS showed compound 4 was consumedcompletely and one main peak with desired m/z was detected. The reactionwas directly purified by prep-HPLC (neutral condition). Compound 5(0.400 g, 594.64 μmol, 31.77% yield) was obtained as a white solid.

Preparation of Compound 6

To a solution of compound 5 (0.200 g, 297.32 μmol, 1.0 eq) in DMF (5 mL)was added HOBt (52.23 mg, 386.51 μmol, 1.3 eq) and DIEA (115.28 mg,891.95 μmol, 155.36 μL, 3.0 eq), MMAE (192.12 mg, 267.59 μmol, 0.9 eq).The mixture was stirred at 20° C. for 16 hr. LC-MS showed compound 5 wasconsumed completely and one main peak with desired m/z was detected. Thereaction was directly purified by prep-HPLC (neutral condition).Compound 6 (0.160 g, 127.84 μmol, 43.00% yield) was obtained as a whitesolid.

Preparation of Compound 7

To a solution of compound 6 (0.160 g, 127.84 μmol, 1.0 eq) in THF (3 mL)and H₂O (3 mL) was added LiOH.H₂O (26.82 mg, 639.21 μmol, 5.0 eq). Themixture was stirred at 20° C. for 1 hr. LC-MS showed compound 6 wasconsumed completely and one main peak with desired m/z was detected. TheTHF was removed under reduced pressure and adjusted the pH=7 by AcOH,the mixture was lyophilizated. Compound 7 (0.130 g, 105.05 μmol, 82.17%yield) was obtained as a white solid.

Preparation of Compound 8

To a solution of compound 7 (36.27 mg, 315.15 μmol, 3.0 eq) in DMA (6mL) and DCM (2 mL) was added EDCI (60.41 mg, 315.15 μmol, 3.0 eq). Themixture was stirred at 15° C. for 3 hr. LC-MS showed compound 7 wasconsumed completely and one main peak with desired m/z was detected. DCMwas removed under reduced pressure. The reaction was directly purifiedby prep-HPLC (neutral condition). Compound 8 (0.095 g, 71.18 μmol,67.76% yield) was obtained as a white solid.

Preparation of BCY8245

To a solution of BCY8234 (66.41 mg, 22.48 μmol, 1.0 eq) in DMA (4 mL)was added DIEA (8.72 mg, 67.44 μmol, 11.75 μL, 3.0 eq) and compound 8(0.030 g, 22.48 μmol, 1.0 eq). The mixture was stirred at 20° C. for 16hr. LC-MS showed BCY8234 was consumed completely and one main peak withdesired m/z or desired mass was detected. The reaction was directlypurified by prep-HPLC (TFA condition). Compound BCY8245 (0.0427 g, 10.16μmol, 45.19% yield, 99.30% purity) was obtained as a white solid.Retention time=11.7 min. Mass found=1043.9 (M/4+H)

Biological Data

Nectin-4 Direct Binding Assay

Affinity of the peptides of the invention for human Nectin-4 (Ki) wasdetermined using a fluorescence polarisation assay, in accordance withthe methods disclosed in WO 2016/067035. Peptides of the invention witha fluorescent tag (either fluorescein, SIGMA or Alexa Fluor488™, FisherScientific) were diluted to 2.5 nM in PBS with 0.01% tween 20 or 50 mMHEPES with 100 mM NaCl and 0.01% tween pH 7.4 (both referred to as assaybuffer). This was combined with a titration of protein in the same assaybuffer as the peptide to give 1 nM peptide in a total volume of 25 μL ina black walled and bottomed low bind low volume 384 well plates,typically 5 μL assay buffer, 10 μL protein then 10 μL fluorescentpeptide. One in two serial dilutions were used to give 12 differentconcentrations with top concentrations ranging from 500 nM for knownhigh affinity binders to 10 μM for low affinity binders and selectivityassays. Measurements were conducted on a BMG PHERAstar FS equipped withan “FP 485 520 520” optic module which excites at 485 nm and detectsparallel and perpendicular emission at 520 nm. The PHERAstar FS was setat 25° C. with 200 flashes per well and a positioning delay of 0.1second, with each well measured at 5 to 10 minute intervals for 60minutes. The gain used for analysis was determined for each tracer atthe end of the 60 minutes where there was no protein in the well. Datawas analysed using Systat Sigmaplot version 12.0. mP values were fit toa user defined quadratic equation to generate a Kd value:f=ymin+(ymax−ymin)/Lig*((x+Lig+Kd)/2−sqrt((((x+Lig+Kd)/2){circumflexover ( )}2)−(Lig*x))). “Lig” was a defined value of the concentration oftracer used.

Nectin-4 Competition Binding Assay

Peptides without a fluorescent tag were tested in competition withACPFGCHTDWSWPIWCA-Sar6-K(FI) (SEQ ID NO: 2) and (Kd=5 nM—determinedusing the protocol above). Peptides were diluted to an appropriateconcentration in assay buffer as described in the direct binding assaywith a maximum of 5% DMSO, then serially diluted 1 in 2. Five μL ofdiluted peptide was added to the plate followed by 10 μL of humanNectin-4, then 10 μL fluorescent peptide added. Measurements wereconducted as for the direct binding assay, however the gain wasdetermined prior to the first measurement. Data analysis was in SystatSigmaplot version 12.0 where the mP values were fit to a user definedcubic equation to generate a Ki value:f=y min+(y max−ymin)/Lig*((Lig*((2*((Klig+Kcomp+Lig+Comp−Prot*c){circumflex over( )}2−3*(Kcomp*(Lig−Prot*c)+Klig*(Comp−Prot*c)+Klig*Kcomp)){circumflexover ( )}0.5*COS(ARCCOS((−2*(Klig+Kcomp+Lig+Comp−Prot*c){circumflex over( )}3+9*(Klig+Kcomp+Lig+Comp−Prot*c)*(Kcomp*(Lig−Prot*c)+Klig*(Comp−Prot*c)+Klig*Kcomp)−27*(−1*Klig*Kcomp*Prot*c))/(2*((((Klig+Kcomp+Lig+Comp−Prot*c){circumflexover( )}2−3*(Kcomp*(Lig−Prot*c)+Klig*(Comp−Prot*c)+Klig*Kcomp)){circumflexover( )}0.5)))/3))−(Klig+Kcomp+Lig+Comp−Prot*c)))/((3*Klig)+((2*((Klig+Kcomp+Lig+Comp−Prot*c){circumflexover( )}2−3*(Kcomp*(Lig−Prot*c)+Klig*(Comp−Prot*c)+Klig*Kcomp)){circumflexover ( )}0.5*COS(ARCCOS((−2*(Klig+Kcomp+Lig+Comp−Prot*c){circumflex over( )}3+9*(Klig+Kcomp+Lig+Comp−Prot*c)*(Kcomp*(Lig−Prot*c)+Klig*(Comp−Prot*c)+Klig*Kcomp)−27*(−1*Klig*Kcomp*Prot*c))/(2*((((Klig+Kcomp+Lig+Comp−Prot*c){circumflexover( )}2−3*(Kcomp*(Lig−Prot*c)+Klig*(Comp−Prot*c)+Klig*Kcomp)){circumflexover ( )}3){circumflex over( )}0.5)))/3))−(Klig+Kcomp+Lig+Comp−Prot*c)))).

“Lig”, “KLig” and “Prot” were all defined values relating to:fluorescent peptide concentration, the Kd of the fluorescent peptide andNectin concentration respectively.

Nectin-4 Biacore SPR Binding Assay

Biacore experiments were performed to determine k_(a) (M⁻¹s⁻¹), k_(d)(s⁻¹), K_(D) (nM) values of monomeric peptides binding to human Necin-4protein (obtained from Charles River).

Human Nectin-4 (residues Gly32-Ser349; NCBI RefSeq: NP_112178.2) with agp67 signal sequence and C-terminal FLAG tag was cloned into pFastbac-1and baculovirus made using standard Bac-to-Bac™ protocols (LifeTechnologies). Sf21 cells at 1×10⁶ ml⁻¹ in Excell-420 medium (Sigma) at27° C. were infected at an MOI of 2 with a P1 virus stock and thesupernatant harvested at 72 hours. The supernatant was batch bound for 1hour at 4° C. with Anti-FLAG M2 affinity agarose resin (Sigma) washed inPBS and the resin subsequently transferred to a column and washedextensively with PBS. The protein was eluted with 100 μg/ml FLAGpeptide. The eluted protein was concentrated to 2 ml and loaded onto anS-200 Superdex column (GE Healthcare) in PBS at 1 ml/min. 2 ml fractionswere collected and the fractions containing Nectin-4 protein wereconcentrated to 16 mg/ml.

The protein was randomly biotinylated in PBS using EZ-Link™Sulfo-NHS-LC-LC-Biotin reagent (Thermo Fisher) as per the manufacturer'ssuggested protocol. The protein was extensively desalted to removeuncoupled biotin using spin columns into PBS.

For analysis of peptide binding, a Biacore 3000 instrument was usedutilising a CM5 chip (GE Healthcare). Streptavidin was immobilized onthe chip using standard amine-coupling chemistry at 25° C. with HBS-N(10 mM HEPES, 0.15 M NaCl, pH 7.4) as the running buffer. Briefly, thecarboxymethyl dextran surface was activated with a 7 minute injection ofa 1:1 ratio of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride (EDC)/0.1 M N-hydroxy succinimide (NHS) at a flow rate of10 μl/min. For capture of streptavidin, the protein was diluted to 0.2mg/ml in 10 mM sodium acetate (pH 4.5) and captured by injecting 120 μlof streptavidin onto the activated chip surface. Residual activatedgroups were blocked with a 7 minute injection of 1 M ethanolamine (pH8.5) and biotinylated Nectin-4 captured to a level of 1,200-1,800 RU.Buffer was changed to PBS/0.05% Tween 20 and a dilution series of thepeptides was prepared in this buffer with a final DMSO concentration of0.5%. The top peptide concentration was 100 nM with 6 further 2-folddilutions. The SPR analysis was run at 25° C. at a flow rate of 50μl/min with 60 seconds association and dissociation between 400 and1,200 seconds depending upon the individual peptide. Data were correctedfor DMSO excluded volume effects. All data were double-referenced forblank injections and reference surface using standard processingprocedures and data processing and kinetic fitting were performed usingScrubber software, version 2.0c (BioLogic Software). Data were fittedusing simple 1:1 binding model allowing for mass transport effects whereappropriate.

Certain peptide ligands of the invention were tested in the abovementioned Nectin-4 binding assays and the results are shown in Table 1:

TABLE 1 Competition Binding Data for Selected Peptide Ligands of theInvention Number of Bicycle No. K_(i) (μM) Experiments BCY8122 0.003 2BCY8126 0.0027 6

Certain bicyclic peptides of the invention were tested in the abovementioned SPR assay and the results are shown in Table 2:

TABLE 2 SPR Data for Selected Peptide Ligands of the Invention Human SPRBicycle No. Kd (nM) n BCY8122 0.89 1 BCY8126 1.07 4 BCY8116 0.372 1 n =mean number of experiments

Certain bicyclic peptides of the invention were conjugated to cytotoxicagents and tested in the above mentioned SPR assay and the results areshown in Table 3:

TABLE 3 SPR Data for Selected BDCs of the Invention Bicyclic DrugConjugate Human SPR (BDC) No. Peptide Kd (nM) BCY8245MMAE-PABC-Cit-Val-Glutaryl- 5.12 (n = 4) BCY8234 BCY8549MMAE-PABC-cyclobutyl-(B- 1.44 (n = 1) Ala)-BCY8234In Vivo Studies

In each of Examples 1 to 5 and 9 the following methodology was adoptedfor each study:

Test and Postitive Control Articles Physical Molecular Storage NumberDescription Weight Purity Condition BCY8245 Lyophilised 4173.85 99.60%stored at −80° C. powder BCY8549 Lyophilised 4157.81 95.70% stored at−80° C. powderExperimental Methods and Procedures(i) Observations

All the procedures related to animal handling, care and the treatment inthe study were performed according to the guidelines approved by theInstitutional Animal Care and Use Committee (IACUC) of WuXi AppTec,following the guidance of the Association for Assessment andAccreditation of Laboratory Animal Care (AAALAC). At the time of routinemonitoring, the animals were daily checked for any effects of tumorgrowth and treatments on normal behavior such as mobility, food andwater consumption (by looking only), body weight gain/loss, eye/hairmatting and any other abnormal effect as stated in the protocol. Deathand observed clinical signs were recorded on the basis of the numbers ofanimals within each subset.

(ii) Tumor Measurements and the Endpoints

The major endpoint was to see if the tumor growth could be delayed ormice could be cured. Tumor volume was measured three times weekly in twodimensions using a caliper, and the volume was expressed in mm³ usingthe formula: V=0.5 a×b² where a and b are the long and short diametersof the tumor, respectively. The tumor size was then used forcalculations of T/C value. The T/C value (in percent) is an indicationof antitumor effectiveness; T and C are the mean volumes of the treatedand control groups, respectively, on a given day.

TGI was calculated for each group using the formula: TGI(%)=[1−(T_(i)−T₀)/(V_(i)−V₀)]×100; T_(i) is the average tumor volume ofa treatment group on a given day, T₀ is the average tumor volume of thetreatment group on the day of treatment start, V_(i) is the averagetumor volume of the vehicle control group on the same day with T_(i),and V₀ is the average tumor volume of the vehicle group on the day oftreatment start.

(iii) Statistical Analysis

Summary statistics, including mean and the standard error of the mean(SEM), are provided for the tumor volume of each group at each timepoint.

Statistical analysis of difference in tumor volume among the groups wasconducted on the data obtained at the best therapeutic time point afterthe final dose.

A one-way ANOVA was performed to compare tumor volume among groups, andwhen a significant F-statistics (a ratio of treatment variance to theerror variance) was obtained, comparisons between groups were carriedout with Games-Howell test. All data were analyzed using GraphPad Prism5.0. P<0.05 was considered to be statistically significant.

Example 1: In Vivo Efficacy Test of BCY8245 in Treatment of NCI-H292Xenograft (Non-Small Cell Lung Cancer (NSCLC) Model) in BALB/c Nude Mice

1. Study Objective

The objective of the research was to evaluate the in vivo anti-tumorefficacy of BCY8245 in treatment of NCI-H292 xenograft model in BALB/cnude mice.

2. Experimental Design

Dosing Dose Volume Dosing Group Treatment n (mg/kg) (μl/g) RouteSchedule 1 Vehicle 3 — 10 iv biw 2 BCY8245 3 1/3/5 10 iv qw 3 BCY8245 33 mg/kg 10 iv qw 4 BCY8245 3 3 mg/kg 10 iv biw 5 BCY8245 3 5 mg/kg 10 ivqw Note: n: animal number; Dosing volume: adjust dosing volume based onbody weight 10 μl/g.3. Materials3.1 Animals and Housing Condition3.1.1. Animals

-   -   Species: Mus Musculus    -   Strain: Balb/c nude    -   Age: 6-8 weeks    -   Sex: female    -   Body weight: 18-22 g    -   Number of animals: 18 mice for BCY8245 plus spare    -   Animal supplier: Shanghai LC Laboratory Animal Co., LTD.        3.1.2. Housing Condition    -   The mice were kept in individual ventilation cages at constant        temperature and humidity with 3 animals in each cage.        -   Temperature: 20˜26° C.        -   Humidity 40-70%.    -   Cages: Made of polycarbonate. The size is 300 mm×180 mm×150 mm.        The bedding material is corn cob, which is changed twice per        week.    -   Diet: Animals had free access to irradiation sterilized dry        granule food during the entire study period.    -   Water: Animals had free access to sterile drinking water.    -   Cage identification: The identification labels for each cage        contained the following information: number of animals, sex,        strain, the date received, treatment, study number, group number        and the starting date of the treatment.    -   Animal identification: Animals were marked by ear coding.        3.2 Test and Positive Control Articles        4. Experimental Methods and Procedures        4.1 Cell Culture

The NCI-H292 tumor cells will be maintained in medium supplemented with10% heat inactivated fetal bovine serum at 37° C. in an atmosphere of 5%CO₂ in air. The tumor cells will be routinely subcultured twice weekly.The cells growing in an exponential growth phase will be harvested andcounted for tumor inoculation.

4.2 Tumor Inoculation

Each mouse will be inoculated subcutaneously at the right flank withNCI-H292 tumor cells (10×10⁶) in 0.2 ml of PBS for tumor development.The animals will be randomized and treatment will be started when theaverage tumor volume reaches approximately 158-406 mm³. The test articleadministration and the animal numbers in each group are shown in thefollowing experimental design table.

4.3 Testing Article Formulation Preparation

Test Con. article (mg/ml) Formulation Vehicle — 25 mM Histidine pH 7 10%sucrose BCY8245 1 Dissolve 1.61 mg BCY8245 with 1.604 ml buffer(vehicle)0.1 Dilute 90 μl 1 mg/ml BCY8245 stock with 810 μl buffer(vehicle) 0.3Dilute 270 μl 1 mg/ml BCY8245 stock with 630 μl buffer(vehicle) 0.5Dilute 450 μl 1 mg/ml BCY8245 stock with 450 μl buffer(vehicle) Vehicle— 25 mM Histidine pH 7 10% sucrose BCY8245 1 Dissolve 10.56 mg BCY8245in 10.518 ml Histidine buffer BCY8245 0.5 Dilute 400 μl 1 mg/ml BCY8245stock with 400 μl Histidine buffer BCY8245 0.3 Dilute 240 μl 1 mg/mlBCY8245 stock with 560 μl Histidine buffer4.4 Sample Collection

At the end of study, the plasma was collected at 5 min, 15 min, 30 min,60 min and 120 min post last dosing.

5. Results

5.1 Tumor Growth Curves

The tumor growth curves are shown in FIGS. 1 and 2.

5.2 Tumor Volume Trace

Mean tumor volume over time in female Balb/c nude mice bearing NCI-H292xenograft is shown in the below Tables:

TABLE 4 Tumor volume trace over time Treatment 0 2 4 7 9 11 14 16Vehicle, qw 410 ± 77 516 ± 69 627 ± 61 931 ± 141 1118 ± 225  1208 ± 2571495 ± 365 1743 ± 419 BCY8245, 404 ± 65 391 ± 42 542 ± 14 721 ± 136 762± 115 607 ± 95 614 ± 89 626 ± 93 1/3/5 mpk, qw Treatment 18 21 23 25 2830 32 35 Vehicle, qw 1950 ± 551 2149 ± 639 BCY8245, 611 ± 93  654 ± 152732 ± 139 755 ± 132 713 ± 114 762 ± 165 968 ± 290 1119 ± 216 1/3/5 mpk,qw

TABLE 5 Tumor volume trace over time Days after the start of treatmentTreatment 0 2 4 7 9 11 14 Vehicle, qw 161 ± 2 270 ± 14 357 ± 14 448 ± 17570 ± 16 720 ± 36 948 ± 61 BCY8245, 160 ± 5 220 ± 11 266 ± 15 218 ± 23167 ± 10 161 ± 36 149 ± 43 3 mpk, qw BCY8245,  162 ± 13 243 ± 19 211 ±12 101 ± 11 100 ± 8  87 ± 7 65 ± 3 3 mpk, biw BCY8245, 160 ± 9 176 ± 7 191 ± 3  105 ± 8  82 ± 3  91 ± 14 83 ± 8 5 mpk, qw5.3 Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for BCY8245 in the NCI-H292 xenograft modelon day 14 was calculated based on tumor volume measurements.

TABLE 6 Tumor growth inhibition analysis Tumor P value Volume T/C^(b)TGI compare Treatment (mm³)^(a) (%) (%) with vehicle Vehicle, qw 2149 ±639  — — — BCY8245, 654 ± 152 30.4 85.7 p < 0.05 1/3/5 mpk, qw ^(a)Mean± SEM. ^(b)Tumor Growth Inhibition is calculated by dividing the groupaverage tumor volume for the treated group by the group average tumorvolume for the control group (T/C).

TABLE 7 Tumor growth inhibition analysis Tumor P value Volume T/C^(b)TGI compared Treatment (mm³)^(a) (%) (%) with vehicle Vehicle, qw 948 ±61 — — — BCY8245, 149 ± 43 15.8 101.4 p < 0.001 3 mpk, qw BCY8245, 65 ±3 6.9 112.2 p < 0.001 3 mpk, biw BCY8245, 83 ± 8 8.8 109.8 p < 0.001 5mpk, qw ^(a)Mean ± SEM. ^(b)Tumor Growth Inhibition is calculated bydividing the group average tumor volume for the treated group by thegroup average tumor volume for the control group (T/C).6. Results Summary and Discussion

In this study, the therapeutic efficacy of BCY8245 in the NCI-H292xenograft model was evaluated. The measured tumor volumes of alltreatment groups at various time points are shown in FIGS. 1 and 2 andTables 4 to 7.

The mean tumor size of vehicle treated mice reached 879 mm³ on day 14.

BCY8245 at 1 mg/kg didn't produce significant antitumor activity, thetest article showed obvious antitumor activity after increasing thedosage to 3 mg/kg from day 7, but the efficacy was not further improvedafter increasing the dosage to 5 mg/kg on day 21. In this study, all ofthe treatment animals showed continued bodyweight loss during the dosingschedule, this may be due to the tumor burden and the toxicity of testarticles.

BCY8245 at 3 mg/kg, qw (TV=149 mm³, TGI=101.4%, p<0.001), 3 mg/kg, biw(TV=65 mm³, TGI=112.2%, p<0.001) and 5 mg/kg, qw (TV=83 mm³, TGI=109.8%,p<0.001) produced significant antitumor activity.

Example 2: In Vivo Efficacy Test of BCY7825, BCY8245, BCY8253, BCY8254and BCY8255 in Treatment of HT-1376 Xenograft (Bladder Cancer Model) inCB17-SCID Mice

1. Study Objective

The objective of the research is to evaluate the in vivo anti-tumorefficacy of test articles in treatment of HT-1376 xenograft in CB17-SCIDmice.

2. Experimental Design

Dosing Dose Volume Dosing Group Treatment n (mg/kg) (μl/g) RouteSchedule 1 Vehicle 3 — 10 iv qw 2 BCY8245 3 1/3^(a) mg/kg    10 iv qw 3Vehicle 5 — 10 iv qw 4 BCY8245 3 3 mg/kg 10 iv qw 5 BCY8245 3 3 mg/kg 10iv biw 6 BCY8245 3 5 mg/kg 10 iv qw ^(a)1 mg/kg for the first week and 3mg/kg for the following 2 weeks3. Materials3.1 Animals and Housing Condition3.1.1. Animals

-   -   Species: Mus Musculus    -   Strain: CB17-SCID    -   Age: 6-8 weeks    -   Sex: female    -   Body weight: 18-22 g    -   Number of animals: 21-41 mice plus spare    -   Animal supplier: Shanghai LC Laboratory Animal Co., LTD.        3.1.2. Housing Condition    -   The mice were kept in individual ventilation cages at constant        temperature and humidity with 3 or 5 animals in each cage.        -   Temperature: 20˜26° C.        -   Humidity 40-70%.    -   Cages: Made of polycarbonate. The size is 300 mm×180 mm×150 mm.        The bedding material is corn cob, which is changed twice per        week.    -   Diet: Animals had free access to irradiation sterilized dry        granule food during the entire study period.    -   Water: Animals had free access to sterile drinking water.    -   Cage identification: The identification labels for each cage        contained the following information: number of animals, sex,        strain, the date received, treatment, study number, group number        and the starting date of the treatment.    -   Animal identification: Animals were marked by ear coding.        4. Experimental Methods and Procedures        4.1 Cell Culture

The HT-1376 tumor cells were maintained in vitro as a monolayer culturein EMEM medium supplemented with 10% heat inactivated fetal bovine serumat 37° C. in an atmosphere of 5% CO₂ in air. The tumor cells wereroutinely subcultured twice weekly by trypsin-EDTA treatment. The cellsgrowing in an exponential growth phase were harvested and counted fortumor inoculation.

4.2 Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank with HT-1376tumor cells (5×10⁶) in 0.2 ml of PBS with matrigel (1:1) for tumordevelopment. Animals were randomized when the average tumor volumereached 153-164 mm³. The test article administration and the animalnumbers in each group were shown in the experimental design table.

4.3 Testing Article Formulation Preparation

Test Con. article (mg/ml) Formulation Vehicle — 25 mM Histidine pH 7 10%sucrose BCY8245 0.1 Dilute 90 μl 1 mg/ml BCY8245 stock with 810 μlbuffer (vehicle) BCY8245 0.3 Dilute 270 μl 1 mg/ml BCY8245 stock with630 μl buffer (vehicle) Vehicle — 25 mM Histidine pH 7 10% sucroseBCY8245 0.5 Dilute 400 μl 1 mg/ml BCY8245 stock with 400 μl Histidinebuffer BCY8245 0.3 Dilute 240 μl 1 mg/ml BCY8245 stock with 560 μlHistidine buffer4.4 Sample Collection

At the end of study, the plasma of group 2 was collected at 5 min, 15min, 30 min, 60 min and 120 min post last dosing. The plasma of group 6was collected at 5 min, 15 min, 30 min, 60 min and 120 min post lastdosing. The tumor of group 6 was collected at 2 h post last dosing. Thetumor of groups 4 and 5 were collected at 2 h post last dosing.

5. Results

5.1 Tumor Growth Curves

Tumor growth curves are shown in FIGS. 3 and 4.

5.2 Tumor Volume Trace

Mean tumor volume overtime in female CB17-SCID mice bearing HT-1376xenograft is shown in Tables 8 and 9.

TABLE 8 Tumor volume trace over time Days after the start of treatmentGr. Treatment 0 2 4 7 9 11 14 16 18 21 1 Vehicle, qw 168 ± 37 220 ± 47274 ± 56 391 ± 73 442 ± 75 503 ± 82 576 ± 84 649 ± 81 801 ± 84 884 ± 812 BCY8245, 164 ± 16 184 ± 12 206 ± 14 265 ± 21 291 ± 10 281 ± 28 335 ±16 354 ± 11 309 ± 19 347 ± 14 1/3 mpk, qw

TABLE 9 Tumor volume trace over time Days after the start of treatmentGr. Treatment 0 2 5 7 9 12 14 3 Vehicle, 153 ± 16 266 ± 30 398 ± 41 529± 56 721 ± 76 908 ± 91 1069 ± 90  qw 4 BCY8245, 153 ± 26 254 ± 53 298 ±69 398 ± 61 468 ± 73 502 ± 67 603 ± 76 3 mpk, qw 5 BCY8245, 154 ± 30 248± 58 203 ± 15 273 ± 45 356 ± 50 391 ± 53 407 ± 53 3 mpk, biw 6 BCY8245,153 ± 15 237 ± 41 228 ± 36 317 ± 31 394 ± 20 438 ± 31 465 ± 33 5 mpk, qw5.3 Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for Test articles in the HT-1376 xenograftmodel was calculated based on tumor volume measurements at day 21 afterthe start of treatment.

TABLE 10 Tumor growth inhibition analysis Tumor P value Volume T/C^(b)TGI compared Gr Treatment (mm³)^(a) (%) (%) with vehicle 1 Vehicle, qw884 ± 81 — — — 2 BCY8245, 347 ± 14 39.2 74.5 p < 0.001 3 mpk, qw^(a)Mean ± SEM. ^(b)Tumor Growth Inhibition is calculated by dividingthe group average tumor volume for the treated group by the groupaverage tumor volume for the control group (T/C).

TABLE 11 Tumor growth inhibition analysis Tumor P value Volume T/C^(b)TGI compared Gr Treatment (mm³)^(a) (%) (%) with vehicle 3 Vehicle, qw1069 ± 90  — — — 4 BCY8245, 603 ± 76 56.4 50.9 p < 0.01  3 mpk, qw 5BCY8245, 407 ± 53 38.1 72.3 p < 0.001 3 mpk, biw 6 BCY8245, 465 ± 3343.5 66.0 p < 0.001 5 mpk, qw ^(a)Mean ± SEM. ^(b)Tumor GrowthInhibition is calculated by dividing the group average tumor volume forthe treated group by the group average tumor volume for the controlgroup (T/C).6. Results Summary and DiscussionGroups 1 and 2

In this study, the therapeutic efficacy of test articles in the HT-1376xenograft model was evaluated. The measured tumor volumes of alltreatment groups at various time points are shown in FIG. 3 and Tables 8and 10.

The mean tumor size of vehicle treated mice reached 884 mm³ on day 21.BCY8245 at 1 mg/kg produced slight antitumor activity, and betterefficacy was found after increasing dosage to 3 mg/kg from day 7.

In this study, some mice treated with the test article at 3 mg/kg showedover 10% bodyweight loss.

Groups 3-6

In this study, the therapeutic efficacy of test articles in the HT-1376xenograft model was evaluated. The measured tumor volumes of alltreatment groups at various time points are shown in FIG. 4 and Tables 9and 11.

BCY8245 at 3 mg/kg, qw (TV=603 mm³, TGI=50.9%, p<0.01), 3 mg/kg, biw(TV=407 mm³, TGI=72.3%, p<0.001) and 5 mg/kg, qw (TV=465 mm³, TGI=66.0%,p<0.001) produced significant antitumor activity.

In this study, BCY8245 at 5 mg/kg qw caused over 10% animal bodyweightloss during the treatment schedule.

Example 3: In Vivo Efficacy Study of BCY8245 in Treatment of Panc2.13Xenograft (Pancreatic Cancer Model) in Balb/c Nude Mice

1. Study Objective

The objective of the research is to evaluate the in vivo anti-tumorefficacy of test articles in treatment of Panc2.13 xenograft in Balb/cnude mice.

2. Experimental Design

Dosing Dose Volume Dosing Group Treatment n (mg/kg) (μl/g) RouteSchedule 1 Vehicle 5 — 10 iv qw 2 BCY8245 3 3 mg/kg 10 iv qw 3 BCY8245 33 mg/kg 10 iv biw 4 BCY8245 3 5 mg/kg 10 iv qw3. Materials3.1 Animals and Housing Condition3.1.1 Animals

-   -   Species: Mus Musculus    -   Strain: Balb/c nude    -   Age: 6-8 weeks    -   Sex: female    -   Body weight: 18-22 g    -   Number of animals: 41 mice plus spare    -   Animal supplier: Shanghai LC Laboratory Animal Co., LTD.        3.1.2. Housing Condition    -   The mice were kept in individual ventilation cages at constant        temperature and humidity with 3 or 5 animals in each cage.        -   Temperature: 20˜26° C.        -   Humidity 40-70%.    -   Cages: Made of polycarbonate. The size is 300 mm×180 mm×150 mm.        The bedding material is corn cob, which is changed twice per        week.    -   Diet: Animals had free access to irradiation sterilized dry        granule food during the entire study period.    -   Water: Animals had free access to sterile drinking water.    -   Cage identification: The identification labels for each cage        contained the following information: number of animals, sex,        strain, the date received, treatment, study number, group number        and the starting date of the treatment.    -   Animal identification: Animals were marked by ear coding.        4. Experimental Methods and Procedures        4.1 Cell Culture

The Panc2.13 tumor cells will be maintained in RMPI1640 mediumsupplemented with 15% heat inactivated fetal bovine serum and 10units/ml human recombinant insulin at 37° C. in an atmosphere of 5% CO₂in air. The tumor cells will be routinely subcultured twice weekly. Thecells growing in an exponential growth phase will be harvested andcounted for tumor inoculation.

4.2 Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank withPanc2.13 tumor cells (5×10⁶) with Matrigel (1:1) in 0.2 ml of PBS fortumor development. 41 animals were randomized when the average tumorvolume reached 149 mm³. The test article administration and the animalnumbers in each group were shown in the experimental design table.

4.3 Testing Article Formulation Preparation

Test Con. article (mg/ml) Formulation Vehicle — 25 mM Histidine pH 7 10%sucrose BCY8245 0.5 Dilute 400 μl 1 mg/ml BCY8245 stock with 400 μlHistidine buffer BCY8245 0.3 Dilute 240 μl 1 mg/ml BCY8245 stock with560 μl Histidine buffer4.4 Sample Collection

At the end of study, the tumor of all groups were collected at 2 h postlast dosing.

5. Results

5.1 Tumor Growth Curves

The tumor growth curve is shown in FIG. 5.

5.2 Tumor Volume Trace

Mean tumor volume over time in female Balb/c nude mice bearing Panc2.13xenograft is shown in Table 12.

TABLE 12 Tumor volume trace over time Days after the start of treatmentGr. Treatment 0 2 4 7 9 11 14 1 Vehicle, 149 ± 12 202 ± 12 240 ± 9  321± 17 410 ± 27 479 ± 32 545 ± 17 qw 2 BCY8245, 149 ± 34 160 ± 33 191 ± 39215 ± 53 242 ± 62 259 ± 59 271 ± 54 3 mpk, qw 3 BCY8245, 148 ± 46 170 ±38 204 ± 57 216 ± 56 236 ± 59 241 ± 60 231 ± 57 3 mpk, biw 4 BCY8245,149 ± 18 180 ± 11 231 ± 33 242 ± 34 248 ± 40 231 ± 37 238 ± 40 5 mpk, qw5.3 Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for Test articles in the Panc2.13 xenograftmodel was calculated based on tumor volume measurements at day 14 afterthe start of treatment.

TABLE 13 Tumor growth inhibition analysis Tumor P value Volume T/C^(b)TGI compared Gr Treatment (mm³)^(a) (%) (%) with vehicle 1 Vehicle, qw545 ± 17 — — — 2 BCY8245, 271 ± 54 49.6 69.2 p < 0.01  3 mpk, qw 3BCY8245, 231 ± 57 42.3 79.1 p < 0.001 3 mpk, biw 4 BCY8245, 238 ± 4043.6 77.5 p < 0.001 5 mpk, qw ^(a)Mean ± SEM. ^(b)Tumor GrowthInhibition is calculated by dividing the group average tumor volume forthe treated group by the group average tumor volume for the controlgroup (T/C).6. Results Summary and Discussion

In this study, the therapeutic efficacy of test articles in the Panc2.13xenograft model was evaluated. The measured tumor volumes of alltreatment groups at various time points are shown in FIG. 5 and Tables12 and 13.

BCY8245 at 3 mg/kg, qw (TV=271 mm³, TGI=69.2%, p<0.01), 3 mg/kg, biw(TV=231 mm³, TGI=79.1%, p<0.001) and 5 mg/kg, qw (TV=238 mm³, TGI=77.5%,p<0.001) produced significant antitumor activity.

Example 4: In Vivo Efficacy Study of BCY8245 in Treatment of MDA-MB-468Xenograft (Breast Cancer Model) in Balb/c Nude Mice

1. Study Objective

The objective of the research is to evaluate the in vivo anti-tumorefficacy of BCY8245 in treatment of MDA-MB-468 xenograft in Balb/c nudemice.

2. Experimental Design

Dosing Dose Volume Dosing Group Treatment n (mg/kg) (μl/g) RouteSchedule 1 Vehicle 5 — 10 iv qw 2 BCY8245 3 3 mg/kg 10 iv qw 3 BCY8245 33 mg/kg 10 iv biw 4 BCY8245 3 5 mg/kg 10 iv qw3. Materials3.1 Animals and Housing Condition3.1.1 Animals

-   -   Species: Mus Musculus    -   Strain: Balb/c nude    -   Age: 6-8 weeks    -   Sex: female    -   Body weight: 18-22 g    -   Number of animals: 41 mice plus spare    -   Animal supplier: Shanghai LC Laboratory Animal Co., LTD.        3.1.2. Housing Condition    -   The mice were kept in individual ventilation cages at constant        temperature and humidity with 3 or 5 animals in each cage.        -   Temperature: 20˜26° C.        -   Humidity 40-70%.    -   Cages: Made of polycarbonate. The size is 300 mm×180 mm×150 mm.        The bedding material is corn cob, which is changed twice per        week.    -   Diet: Animals had free access to irradiation sterilized dry        granule food during the entire study period.    -   Water: Animals had free access to sterile drinking water.    -   Cage identification: The identification labels for each cage        contained the following information: number of animals, sex,        strain, the date received, treatment, study number, group number        and the starting date of the treatment.    -   Animal identification: Animals were marked by ear coding.        4. Experimental Methods and Procedures        4.1 Cell Culture

The tumor cells were maintained in Leibovitz's L-15 medium supplementedwith 10% heat inactivated fetal bovine serum at 37° C. in an atmosphereof 5% CO₂ in air. The tumor cells were routinely subcultured twiceweekly. The cells growing in an exponential growth phase were harvestedand counted for tumor inoculation.

4.2 Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank withMDA-MB-468 tumor cells (10×10⁶) in 0.2 ml of PBS supplemented with 50%matrigel for tumor development. 41 animals were randomized when theaverage tumor volume reached 196 mm³. The test article administrationand the animal numbers in each group were shown in the experimentaldesign table.

4.3 Testing Article Formulation Preparation

Test Con. article (mg/ml) Formulation Vehicle — 25 mM Histidine pH 7 10%sucrose BCY8245 1 Dissolve 10.56 mg BCY8245 in 10.518 ml Histidinebuffer BCY8245 0.5 Dilute 400 μl 1 mg/ml BCY8245 stock with 400 μlHistidine buffer BCY8245 0.3 Dilute 240 μl 1 mg/ml BCY8245 stock with560 μl Histidine buffer4.4 Sample Collection

At the day 21 of study, the plasma of group 2 was collected at 5 min, 15min, 30 min, 60 min and 120 min post last dosing. The tumors of groups 1and 3 were collected at 2 h post last dosing. The animals in group 4were kept running for another 21 days without any dosing, and the tumorsof these groups were collected on day 42.

5. Results

5.1 Tumor Growth Curves

The tumor growth curve is shown in FIG. 6.

5.2 Tumor Volume Trace

Mean tumor volume over time in female Balb/c nude mice bearingMDA-MB-468 xenograft is shown in Tables 14 and 15.

TABLE 14 Tumor volume trace over time (Day 0 to day 21) Days after thestart of treatment Gr. Treatment 0 2 4 7 9 11 14 16 18 21 1 Vehicle, qw199 ± 6  217 ± 9  235 ± 15 274 ± 14 291 ± 14 314 ± 20 348 ± 24  374 ±33  398 ± 39  447 ± 39 2 BCY8245, 194 ± 12 192 ± 26 184 ± 20 131 ± 20113 ± 17 104 ± 13 94 ± 25 81 ± 23 87 ± 23  85 ± 31 3 mpk, qw 3 BCY8245,195 ± 33 193 ± 27 154 ± 20 103 ± 20  83 ± 16  67 ± 14 49 ± 11 45 ± 14 32± 12 22 ± 4 3 mpk, biw 4 BCY8245, 199 ± 28 193 ± 11 135 ± 4  98 ± 5 58 ±5 49 ± 5 47 ± 2  37 ± 4  35 ± 3  29 ± 3 5 mpk, qw

TABLE 15 Tumor volume trace over time (Day 23 to day 42) Days after thestart of treatment Gr. Treatment 23 25 28 32 35 39 42 4 BCY8245, 35 ± 548 ± 5 37 ± 7 28 ± 6 24 ± 4 28 ± 6 26 ± 6 5 mpk, qw5.3 Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for test articles in the MDA-MB-468xenograft model was calculated based on tumor volume measurements at day21 after the start of the treatment.

TABLE 16 Tumor growth inhibition analysis Tumor T/C^(b) TGI P value GrTreatment Volume (%) (%) with 1 Vehicle, qw 447 ± 39 — — — 2 BCY8245, 85 ± 31 18.9 144.2 p < 0.001 3 mpk, qw 3 BCY8245, 22 ± 4 4.9 169.8 p <0.001 3 mpk, biw 4 BCY8245, 29 ± 3 6.6 168.4 p < 0.001 5 mpk, qw^(a)Mean ± SEM. ^(b)Tumor Growth Inhibition is calculated by dividingthe group average tumor volume for the treated group by the groupaverage tumor volume for the control group (T/C).6. Results Summary and Discussion

In this study, the therapeutic efficacy of test articles in theMDA-MB-468 xenograft model was evaluated. The measured tumor volumes ofall treatment groups at various time points are shown in FIG. 6 andTables 14 to 16.

BCY8245 at 3 mg/kg, qw (TV=85 mm³, TGI=144.2%, p<0.001), 3 mg/kg, biw(TV=22 mm³, TGI=169.8%, p<0.001) and 5 mg/kg, qw (TV=29 mm³, TGI=168.4%,p<0.001) produced significant anti-tumor antitumor activity in dose ordose-frequency dependent manner.

The dosing of 5 mg/kg groups were suspended from day 21, the tumorsdidn't show any relapse during extra 3 weeks' monitoring schedule.

Example 5: In Vivo Efficacy Test of BCY8549 in Treatment of NCI-H292Xenograft (Non-Small Cell Lung Cancer (NSCLC) Model) in BALB/c Nude Mice

1. Study Objective

The objective of the research is to evaluate the in vivo anti-tumorefficacy of BCY8549 in treatment of NCI-H292 xenograft in Balb/c nudemice.

2. Experimental Design

Dose Dosing Dosing Group Treatment n (mg/kg) Volume (μl/g) RouteSchedule 1 Vehicle 4 — 10 iv qw 2 BCY8549 3 3 10 iv qw3. Materials3.1 Animals and Housing Condition3.1.1. Animals

-   -   Species: Mus Musculus    -   Strain: Balb/c nude    -   Age: 6-8 weeks    -   Sex: female    -   Body weight: 18-22 g    -   Number of animals: 43 mice plus spare    -   Animal supplier: Shanghai Lingchang Biotechnology Experimental        Animal Co. Ltd        3.1.2. Housing Condition    -   The mice were kept in individual ventilation cages at constant        temperature and humidity with 3 or 4 animals in each cage.        -   Temperature: 20˜26° C.        -   Humidity 40-70%.    -   Cages: Made of polycarbonate. The size is 300 mm×180 mm×150 mm.        The bedding material is corn cob, which is changed twice per        week.    -   Diet: Animals had free access to irradiation sterilized dry        granule food during the entire study period.    -   Water: Animals had free access to sterile drinking water.    -   Cage identification: The identification labels for each cage        contained the following information: number of animals, sex,        strain, the date received, treatment, study number, group number        and the starting date of the treatment.    -   Animal identification: Animals were marked by ear coding.        4. Experimental Methods and Procedures        4.1 Cell Culture

The NCI-H292 tumor cells were maintained in vitro as a monolayer culturein RPMI-1640 medium supplemented with 10% heat inactivated fetal bovineserum at 37° C. in an atmosphere of 5% CO₂ in air. The tumor cells wereroutinely subcultured twice weekly by trypsin-EDTA treatment. The cellsgrowing in an exponential growth phase were harvested and counted fortumor inoculation.

4.2 Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank withNCI-H292 tumor cells (10×10⁶) in 0.2 ml of PBS for tumor development. 43animals were randomized when the average tumor volume reached 168 mm³.The test article administration and the animal numbers in each groupwere shown in the experimental design table.

Testing Article Formulation Preparation

Con. Treatment (mg/ml) Formulation Vehicle — 25 mM Histidine pH 7 10%sucrose BCY8549 1 Dissolved 2 mg BCY8549 with 1914 μl vehicle bufferBCY8549 0.3 Dilute 240 μl 1 mg/ml BCY8549 stock with 560 μl vehiclebuffer4.4 Sample Collection

At the end of study, the plasma of group 2 mice was collected at 5 min,15 min, 30 min, 1 h and 2 h post the last dosing.

5. Results

5.1 Tumor Growth Curves

The tumor growth curve is shown in FIG. 7.

5.2 Tumor Volume Trace

Mean tumor volume over time in female Balb/c nude mice bearing NCI-H292xenograft is shown in Table 17.

TABLE 17 Tumor volume trace over time Days after the start of treatmentGr. Treatment 0 2 4 7 9 11 14 1 Vehicle, qw 168 ± 16 297 ± 48 362 ± 58460 ± 62 548 ± 69 697 ± 102  843 ± 152 2 BCY8549, 168 ± 30 187 ± 36 164± 31 205 ± 50 234 ± 57 240 ± 98  251 ± 66 3 mpk, qw 3 BCY8550, 167 ± 18208 ± 21 237 ± 16 324 ± 35 421 ± 35 489 ± 44  545 ± 77 3 mpk, qw 4BCY8783, 167 ± 28 182 ± 27 161 ± 40 137 ± 19 135 ± 22 97 ± 20  97 ± 19 3mpk, qw 5 BCY8784, 167 ± 36 165 ± 28 111 ± 19 121 ± 12 123 ± 8  99 ± 1094 ± 7 3 mpk, qw5.3 Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for test articles in the NCI-H292 xenograftmodel was calculated based on tumor volume measurements at day 14 afterthe start of treatment.

TABLE 18 Tumor growth inhibition analysis Tumor Volume T/C^(b) TGI GrTreatment (mm³)^(a) (%) (%) P value 1 Vehicle, qw 843 ± 152 — — — 2BCY8549, 251 ± 66  29.8 87.7 p < 0.05 3 mpk, qw ^(a)Mean ± SEM.^(b)Tumor Growth Inhibition is calculated by dividing the group averagetumor volume for the treated group by the group average tumor volume forthe control group (T/C).6. Results Summary and Discussion

In this study, the therapeutic efficacy of BCYs in the NCI-H292xenograft model was evaluated. The measured tumor volume of alltreatment groups at various time points are shown in FIG. 7 and Tables17 and 18.

The mean tumor size of vehicle treated mice reached 843 mm³ on day 14.BCY8549 at 3 mg/kg showed significant anti-tumor activity. In thisstudy, all mice maintained bodyweight well.

Example 6: Investigation of Association Between Copy Number Variation(CNV) and Gene Expression for Nectin-4 from Multiple Tumour Types

Methods

1. Select all studies in cBioPortal (http:/www.cbioportal.org/) andsearch for NECTIN4.

-   -   (a) Remove provisional studies.    -   (b) Deselect studies with overlapping samples to prevent sample        bias (based on warning in cBioPortal)—always keep PanCancer        study if this is an option.    -   (c) Studies selected for analysis (Table 19).

TABLE 19 Studies analysed from cBioPortal and units in study Study NameUnits Breast Cancer (METABRIC, Nature mRNA expression (microarray) 2012& Nat Commun 2016) Breast Invasive Carcinoma (TCGA, mRNA ExpressionBatch PanCancer Atlas) Normalized/Merged from Illumina HiSeq_RNASeqV2syn4976369 Uterine Corpus Endometrial Carcinoma mRNA Expression BatchNormalized/Merged (TCGA, PanCancer Atlas) from Illumina HiSeq_RNASeqV2syn4976369 Bladder Urothelial Carcinoma (TCGA, RSEM (Batch normalizedfrom Illumina PanCancer Atlas) HiSeq_RNASeqV2) Lung Adenocarcinoma(TCGA, mRNA Expression, RSEM (Batch normalized PanCancer Atlas) fromHiSeq_RNASeqV2) Cervical Squamous Cell Carcinoma RSEM (Batch normalizedfrom Illumina (TCGA, PanCancer Atlas) HiSeq_RNASeqV2) Lung Squamous CellCarcinoma (TCGA, mRNA Expression Batch Normalized/Merged PanCancerAtlas) from Illumina HiSeq_RNASeqV2 syn4976369 Head and Neck SquamousCell mRNA Expression, RSEM (Batch normalized Carcinoma (TCGA, PanCancerAtlas) from Illumina HiSeq_RNASeqV2) Pancreatic Adenocarcinoma (TCGA,mRNA Expression Batch Normalized/Merged PanCancer Atlas) from IlluminaHiSeq_RNASeqV2 syn4976369 Thyroid Carcinoma (TCGA, PanCancer mRNAExpression Batch Normalized/Merged Atlas) from Illumina HiSeq_RNASeqV2syn4976369 Colon Adenocarcinoma (TCGA, RSEM (Batch normalized fromIllumina PanCancer Atlas) HiSeq_RNASeqV2) Thymoma (TCGA, PanCancerAtlas) mRNA Expression Batch Normalized/Merged from IlluminaHiSeq_RNASeqV2 syn4976369 Sarcoma (TCGA, PanCancer Atlas) mRNAExpression Batch Normalized/Merged from Illumina HiSeq_RNASeqV2syn4976369 Stomach Adenocarcinoma (TCGA, mRNA Expression BatchNormalized/Merged PanCancer Atlas) from Illumina HiSeq_RNASeqV2syn4976369 Prostate Adenocarcinoma (TCGA, mRNA Expression, RSEM (Batchnormalized PanCancer Atlas) from Illumina HiSeq_RNASeqV2) KidneyChromophobe (TCGA, mRNA Expression, RSEM (Batch normalized PanCancerAtlas) from Illumina HiSeq_RNASeqV2) Rectum Adenocarcinoma (TCGA, mRNAExpression Batch Normalized/Merged PanCancer Atlas) from IlluminaHiSeq_RNASeqV2 syn4976369 Metastatic Prostate Cancer, SU2C/PCF mRNAexpression/capture (RNA Seq Dream Team (Robinson et al., Cell 2015)RPKM) Pheochromocytoma and Paraganglioma mRNA Expression BatchNormalized/Merged (TCGA, PanCancer Atlas) from Illumina HiSeq_RNASeqV2syn4976369 Kidney Renal Clear Cell Carcinoma mRNA Expression, RSEM(Batch normalized (TCGA, PanCancer Atlas) from Illumina HiSeq_RNASeqV2)Prostate Adenocarcinoma (Fred mRNA expression Hutchinson CRC, Nat Med2016) Colorectal Adenocarcinoma (TCGA, RNA Seq RPKM Nature 2012) OvarianSerous Cystadenocarcinoma mRNA Expression Batch (TCGA, PanCancer Atlas)Normalized/Merged from Illumina HiSeq_RNASeqV2 syn4976369 Kidney RenalPapillary Cell Carcinoma mRNA Expression, RSEM (Batch normalized (TCGA,PanCancer Atlas) from Illumina HiSeq_RNASeqV2) Brain Lower Grade Glioma(TCGA, RSEM (Batch normalized from Illumina PanCancer Atlas)HiSeq_RNASeqV2) Esophageal Adenocarcinoma (TCGA, RSEM (Batch normalizedfrom Illumina PanCancer Atlas) HiSeq_RNASeqV2) Adrenocortical Carcinoma(TCGA, RSEM (Batch normalized from Illumina PanCancer Atlas)HiSeq_RNASeqV2) Glioblastoma Multiforme (TCGA, mRNA Expression, RSEM(Batch normalized PanCancer Atlas) from Illumina HiSeq_RNASeqV2)Prostate Adenocarcinoma (MSKCC, mRNA Expression Cancer Cell 2010)Uterine Carcinosarcoma (TCGA, mRNA Expression Batch Normalized/MergedPanCancer Atlas) from Illumina HiSeq_RNASeqV2 syn4976369 Acute MyeloidLeukemia (TCGA, mRNA Expression, RSEM (Batch normalized PanCancer Atlas)from Illumina HiSeq_RNASeqV2) Skin Cutaneous Melanoma (TCGA, mRNAExpression Batch Normalized/Merged PanCancer Atlas) from IlluminaHiSeq_RNASeqV2 syn4976369 Mesothelioma (TCGA, PanCancer Atlas) mRNAExpression Batch Normalized/Merged from Illumina HiSeq_RNASeqV2syn4976369 Cholangiocarcinoma (TCGA, PanCancer RSEM (Batch normalizedfrom Illumina Atlas) HiSeq_RNASeqV2) Pediatric Acute Lymphoid Leukemia -NECTIN4: mRNA expression (RNA-Seq Phase II (TARGET, 2018) RPKM) DiffuseLarge B-Cell Lymphoma (TCGA, mRNA Expression, RSEM (Batch normalizedPanCancer Atlas) from lllumina HiSeq_RNASeqV2) Cancer Cell LineEncyclopedia mRNA expression (microarray) (Novartis/Broad, Nature 2012)Uveal Melanoma (TCGA, PanCancer mRNA Expression Batch Normalized/MergedAtlas) from Illumina HiSeq_RNASeqV2 syn4976369 Pediatric Wilms' Tumor(TARGET, 2018) NECTIN4: mRNA expression (RNA-Seq RPKM) Testicular GermCell Tumors (TCGA, mRNA Expression Batch Normalized/Merged PanCancerAtlas) from Illumina HiSeq_RNASeqV2 syn4976369 Liver HepatocellularCarcinoma (TCGA, NECTIN4: mRNA Expression, RSEM (Batch PanCancer Atlas)normalized from Illumina HiSeq_RNASeqV2)2. Export CNV and RNA expression data from cBioPortal.3. Test if CNVs are statistically significantly associated with changesin mRNA expression for Nectin-4 (log 2 not applied).

-   -   (a) Run non-parametric Kruskal-Wallis test in GraphPad Prism        (7.04) and R/R studio (threshold for significance: p<0.01).        -   (i) GraphPad Prism: set up column table, run non-parametric            test with no matching or pairing and do not assume Gaussian            distribution.        -   (ii) Packages used in R:            -   1. XLConnect            -   2. dplyr            -   3. Kruskal-Wallis Rank Sum Test: Kruskal.test.                4. Adjust for multiple comparisons (include all possible                comparisons even if n=1 within a group) in R/Rstudio                using Dunn's test (threshold for significance: p<0.025).    -   (a) dunn.test with multiple comparison method=“bonferonni”.        Results

The results are shown in Table 20 below. Across 41 publicly availableTCGA datasets that report both tumor CNV and mRNA gene expression datafor Nectin-4, there are many indications where cases have been reportedwith either Nectin-4 copy number gains (2-3 copies) or amplifications(>3 copies). In addition, separate cases have been demonstrated to haveshallow deletions (<2 copies) with rare reports of tumors containingdeep deletions consistent with greater than 1 copy loss or biallelicNectin-4 loss. Indications where amplifications were detected mostfrequently were breast cancer (10-22%), bladder cancer (20%), lungcancer (5-7%) and hepatocellular carcinoma (8%). Indications with mostfrequent copy number losses were kidney chromophobe (77%), renal clearcell carcinoma (RCC) (6.5%), sarcoma (10%), colon cancer (10%), head andneck cancer (7%) and lung squamous cancer. These data indicate thatthere are a range of CNV within and across tumor indications and adiversity of copy number patterns across different indications.

In addition to CNVs within the TCGA dataset the median Nectin-4 mRNAexpression level per indication covers approximately 2¹⁰ range.Therefore, given the range of Nectin-4 mRNA expression levels and theCNVs observed across and within tumor types statistical testing wasperformed to identify potential associations between Nectin-4 mRNAlevels and Nectin-4 CNVs within individual TCGA datasets/indications.Tumors per indication were allocated to 1 of 5 classes:

-   -   a) Deep deletion;    -   b) Shallow deletion;    -   c) Diploid;    -   d) Gain; or    -   e) Amplification.

Kruskall-Wallis testing was then performed to detect if thedistributions of mRNA expression values per classes differed betweenclasses (P<0.01). For those TCGA data sets with P<0.01 and to identifywhich classes were different to one another post hoc testing wasperformed by calculating Z-statistics with adjusted P-values calculated(Bonferonni). For simplicity of interpretation pair-wise comparisons vs.diploid per indication were reviewed (although all pair-wise P-valueswere calculated). 18/41 TCGA studies met Kruskall-Wallis P<0.01 &Bonferonni P<0.025 for Gain vs. Diploid and/or Amplification vs. Diploidcomparisons indicating an association of increased Nectin-4 mRNAexpression with increased Nectin-4 copy number. These 18 studiesrepresented 14 independent tumor histologies:

-   -   breast, uterine, bladder, lung adenocarcinoma, lung squamous,        cervical, head and neck, pancreatic, thyroid, colorectal,        thymoma, sarcoma, renal clear cell carcinoma (RCC) and stomach.

In addition, 6 studies have decreased mRNA expression associated withcopy number loss. Four of these six studies not only showed anassociation between CNV loss and reduced expression, but also reportedCNV gains associated with high expression:

-   -   stomach, lung squamous, colon and thyroid.

Whereas two indications, kidney chromophobe and prostate cancer onlyreported associations with CNV loss and low transcript abundance.Additionally, there was a separate prostate cancer study (MetastaticProstate Cancer, SU2C/PCF Dream Team (Robinson et al., Cell 2015)) thatshowed copy number gains associated with high expression (relative todiploid).

These observations of tumor CNV loss and gain with mRNA expressionlevels may represent the mechanism behind Nectin-4 tumor proteinexpression in those indications where such associations were observed.Clearly there are indications where CNVs do not appear to impact mRNAexpression levels in a predictable pattern such as hepatocellularcarcinoma. In vivo preclinical efficacy with certain Nectin-4 bicyclicdrug conjugates of the invention has been demonstrated to correlate withNectin-4 protein expression as measured by IHC. Therefore, if tumorNectin-4 CNVs associate with mRNA levels and predict protein expressionlevels it is formally possible that patients with tumors containing copynumber increases (gain or amplification) may be more likely to respondto Nectin-4 bicyclic drug conjugates of the invention. If patients couldbe identified with increased CNV in Nectin-4 then this information couldbe used to select patients for treatment with Nectin-4 bicyclic drugconjugates of the invention.

TABLE 20 Results of Investigation of Association between Copy NumberVariation (CNV) and gene expression for Nectin-4 Pairwise Comparison, Zstatistic Number of samples/group (n = X) Kruskal-Wallis Test (adjustedp-value), Bonferonni Deep Kruskal-Wallis Deep Deletion - Diploid -Amplification - Study name Units deletion Shallow deletion Diploid GainAmplification Statistic p-value Diploid Shallow deletion Diploid - GainDiploid Breast Cancer mRNA 0 11 745 706 404 380.4 <2.2e−16 N/A 0.568782−11.89096 18.85085 (METABRIC, expression (1.0000) (0.0000)* (0.0000)*Nature 2012 & (microarray) Nat Commun 2016) Breast mRNA 0 13 244 640 97219.5 <2.2e−16 N/A 1.186089 −12.30176 12.07432 Invasive Expression(0.7068) (0.0000)* (0.0000)* Carcinoma Batch (TCGA, Normalized/MergedPanCancer from Atlas) Illumina HiSeq_RNASeq V2 syn4976369 Uterine CorpusmRNA 0 5 274 210 18 76.392 <2.2e−16 N/A 1.130854 −7.274308 5.601260Endometrial Expression (0.7743) (0.0000*) (0.0000*) Carcinoma Batch(TCGA, Normalized/Merged PanCancer from Atlas) IIlumina HiSeq_RNASeq V2syn4976369 Bladder RSEM (Batch 0 16 171 145 70 67.078 1.80E−14 N/A0.060907 −3.323839 8.054269 Urothelial normalized from (1.0000)(0.0027)* (0.0000)* Carcinoma IIlumina (TCGA, HiSeq_RNASeq PanCancer V2)Atlas) Lung mRNA 0 9 129 332 33 59.578 7.24E−13 N/A 0.237200 −6.2441566.247228 Adenocarcinoma Expression, (1.0000) (0.0000)* (0.0000)* (TCGA,RSEM (Batch PanCancer normalized from Atlas) IIlumina HiSeq_RNASeq V2)Cervical RSEM (Batch 0 7 115 147 6 51.372 4.08E−11 N/A 1.093749−6.170067 3.815296 Squamous normalized from (0.8222) (0.0000)* (0.0004)*Cell IIlumina Carcinoma HiSeq_RNASeq (TCGA, V2) PanCancer Atlas) LungmRNA 0 22 199 222 23 42.128 3.77E−09 N/A 2.819759 −3.034709 4.860629Squamous Expression (0.0144)* (0.0072)* (0.0000)* Cell Batch CarcinomaNormalized/Merged (TCGA, from PanCancer IIlumina Atlas) HiSeq_RNASeq V2syn4976369 Head and mRNA 0 32 330 122 4 37.81 3.10E−08 N/A 1.736867−4.848550 3.033083 Neck Expression, (0.2472) (0.0000)* (0.0073)*Squamous RSEM (Batch Cell normalized from Carcinoma IIlumina (TCGA,HiSeq_RNASeq PanCancer V2) Atlas) Pancreatic mRNA 0 7 105 50 6 36.8634.92E−08 N/A 1.333193 −4.388701 4.166401 Adenocarcinoma Expression(0.5474) (0.0000)* (0.0001)* (TCGA, Batch PanCancer Normalized/MergedAtlas) from IIlumina HiSeq_RNASeq V2 syn4976369 Thyroid mRNA 0 3 451 260 31.882 1.19E−07 N/A 2.486724 −5.021279 N/A Carcinoma Expression(0.0193)* (0.0000)* (TCGA, Batch PanCancer Normalized/Merged Atlas) fromIIlumina HiSeq_RNASeq V2 syn4976369 Colon RSEM (Batch 0 40 266 80 231.309 7.32E−07 N/A 3.811621 −2.987223 1.759508 Adenocarcinomanormalized from (0.0004)* (0.0084)* (0.2355) (TCGA, IIlumina PanCancerHiSeq_RNASeq Atlas) V2) Thymoma mRNA 0 0 95 22 2 26.213 2.03E−06 N/A N/A−4.962115 1.567541 (TCGA, Expression (0.0000)* (0.1755) PanCancer BatchAtlas) Normalized/Merged from IIlumina HiSeq_RNASeq V2 syn4976369Sarcoma mRNA 0 22 120 74 14 26.831 6.39E−06 N/A −0.410850 −4.5820473.106262 (TCGA, Expression (1.0000) (0.0000)* (0.0057)* PanCancer BatchAtlas) Normalized/Merged from IIlumina HiSeq_RNASeq V2 syn4976369Stomach mRNA 0 11 253 134 9 19.096 0.0002611 N/A 2.835658 −2.921683−0.265333 Adenocarcinoma Expression (0.0137)* (0.0104)* (1.0000) (TCGA,Batch PanCancer Normalized/Merged Atlas) from IIlumina HiSeq_RNASeq V2syn4976369 Prostate mRNA 3 15 437 29 3 19.125 0.0007426 −2.5327343.202764 −1.351661 0.509151 Adenocarcinoma Expression, (0.0566)(0.0068)* (0.8824) (1.0000) (TCGA, RSEM (Batch PanCancer normalized fromAtlas) Illumina HiSeq_RNASeq V2) Kidney mRNA 0 50 14 1 0 13.8510.0009823 N/A 3.609735 −0.058395 N/A Chromophobe Expression, (0.0005)*(1.0000) (TCGA, RSEM (Batch PanCancer normalized from Atlas) IlluminaHiSeq_RNASeq V2) Rectum mRNA 0 11 91 33 1 14.056 0.00283 N/A 1.050760−2.951363 1.809506 Adenocarcinoma Expression (0.8801) (0.0095)* (0.2111)(TCGA, Batch PanCancer Normalized/Merged Atlas) from IlluminaHiSeq_RNASeq V2 syn4976369 Metastatic mRNA 0 3 75 37 2 12.336 0.006317N/A 0.040058 −3.420479 −0.362109 Prostate expression/ (1.0000) (0.0019)*(1.0000) Cancer, capture (RNA SU2C/PCF Seq RPKM) Dream Team (Robinson etal., Cell 2015) Pheochromocytoma mRNA 0 14 123 19 5 11.573 0.008998 N/A−1.271308 −2.597791 2.201970 and Expression (0.6109) (0.0281) (0.0830)Paraganglioma Batch (TCGA, Normalized/Merged PanCancer from Atlas)IIlumina HiSeq_RNASeq V2 syn4976369 Kidney Renal mRNA 0 22 297 32 111.314 0.01014 N/A −0.748380 −2.996852 1.502464 Clear Cell Expression,(1.0000) (0.0082)* (0.3989)* Carcinoma RSEM (Batch (TCGA, normalizedfrom PanCancer IIlumina Atlas) HiSeq_RNASeq V2) Prostate mRNA 0 1 59 667 9.8842 0.01958 N/A 0.677737 −0.409530 3.028793 Adenocarcinomaexpression (1.0000) (1.0000) (0.0074)* (Fred Hutchinson CRC, Nat Med2016) Colorectal RNA Seq RPKM 0 4 153 34 2 9.4054 0.02 N/A 0.062894−1.860678 2.514653 Adenocarcinoma (1.0000) (0.1884) (0.0357) (TCGA,Nature 2012) Ovarian mRNA 0 7 75 117 2 9.3101 0.02544 N/A 1.589035−2.168253 0.062706 Serous Expression (0.3362) (0.0904) (1.0000)Cystadenocarcinoma Batch (TCGA, Normalized/Merged PanCancer from Atlas)Illumina HiSeq_RNASeq V2 syn4976369 Kidney Renal mRNA 1 19 239 15 09.1134 0.02782 1.607764 −0.569938 −2.552083 N/A Papillary CellExpression, (0.3237) (1.0000) (0.0321) Carcinoma RSEM (Batch (TCGA,normalized from PanCancer Illumina Atlas) HiSeq_RNASeq V2) Brain LowerRSEM (Batch 0 11 462 32 2 4.769 0.1895 N/A 0.462462 −1.718955 1.261960Grade Glioma normalized from (1.0000) (0.2569) (0.6209) (TCGA, IlluminaPanCancer HiSeq_RNASeq Atlas) V2) Esophageal RSEM (Batch 0 8 78 86 94.267 0.234 N/A 0.747441 −0.768855 1.756911 Adenocarcinoma normalizedfrom (1.0000) (1.0000) (0.2368) (TCGA, Illumina PanCancer HiSeq_RNASeqAtlas) V2) Adrenocortical RSEM (Batch 0 9 54 11 2 4.0298 0.2583 N/A0.157281 −0.131234 1.984800 Carcinoma normalized from (1.0000) (1.0000)(0.1415) (TCGA, Illumina PanCancer HiSeq_RNASeq Atlas) V2) GlioblastomamRNA 0 3 115 27 0 2.6252 0.2691 N/A 0.180194 −1.593755 N/A MultiformeExpression, (1.0000) (0.1665) (TCGA, RSEM (Batch PanCancer normalizedfrom Atlas) Illumina HiSeq_RNASeq V2) Prostate mRNA 0 3 78 4 0 2.1810.3361 N/A 1.423206 0.454433 N/A Adenocarcinoma Expression (0.2320)(0.9743) (MSKCC, Cancer Cell 2010) Uterine mRNA 0 4 14 37 1 3.308 0.3465N/A 0.104285 −0.539065 1.764353 Carcinosarcoma Expression (1.0000)(1.0000) (0.2330) (TCGA, Batch PanCancer Normalized/Merged Atlas) fromIllumina HiSeq_RNASeq V2 syn4976369 Acute Myeloid mRNA 0 0 163 2 00.82638 0.3633 N/A N/A 0.909052 N/A Leukemia Expression, (0.1817) (TCGA,RSEM (Batch PanCancer normalized from Atlas) Illumina HiSeq_RNASeq V2)Skin mRNA 1 19 146 189 8 3.6483 0.4557 −0.898187 −1.116994 −1.2353170.900287 Cutaneous Expression (1.0000) (1.0000) (1.0000) (1.0000)Melanoma Batch (TCGA, Normalized/Merged PanCancer from Atlas) IlluminaHiSeq_RNASeq V2 syn4976369 Mesothelioma mRNA 0 2 56 23 1 2.3747 0.4984N/A −1.426445 0.418206 0.143440 (TCGA, Expression (0.4612) (1.0000)(1.0000) PanCancer Batch Atlas) Normalized/Merged from IlluminaHiSeq_RNASeq V2 syn4976369 Cholangiocarcinoma RSEM (Batch 0 0 13 18 51.3058 0.5205 N/A N/A −0.653051 1.121055 (TCGA, normalized from (0.7706)(0.3934) Pan Cancer Illumina Atlas) HiSeq_RNASeq V2) Pediatric AcuteNECTIN4: 0 4 66 10 1 2.2337 0.5253 N/A 0.133399 −0.504875 −1.375728Lymphoid mRNA (1.0000) (1.0000) (0.5067) Leukemia - expression Phase II(RNA-Seq (TARGET, RPKM) 2018) Diffuse Large mRNA 0 2 25 9 1 1.49390.6837 N/A 0.374642 1.170326 −0.405844 B-Cell Expression, (1.0000)(0.7256) (1.0000) Lymphoma RSEM (Batch (TCGA, normalized from PanCancerIllumina Atlas) HiSeq_RNASeq V2) Cancer Cell mRNA 1 112 396 319 491.9013 0.7539 −0.398204 0.562427 −0.847920 −0.379251 Line expression(1.0000) (1.0000) (1.0000) (1.0000) Encyclopedia (microarray)(Novartis/Broad, Nature 2012) Uveal mRNA 0 0 72 8 0 0.067914 0.7944 N/AN/A −0.260603 N/A Melanoma Expression (0.3972) (TCGA, Batch PanCancerNormalized/Merged Atlas) from Illumina HiSeq_RNASeq V2 syn4976369Pediatric NECTIN4: 0 1 50 46 4 0.78538 0.853 N/A 0.165021 −0.815915−0.090701 Wilms' Tumor mRNA (1.0000) (1.0000) (1.0000) (TARGET,expression 2018) (RNA-Seq RPKM) Testicular mRNA 0 1 76 67 0 0.142790.9311 N/A −0.366969 0.061216 N/A Germ Cell Expression (1.0000) (1.0000)Tumors Batch (TCGA, Normalized/Merged PanCancer from Atlas) IlluminaHiSeq_RNASeq V2 syn4976369 Liver NECTIN4: 0 1 89 224 34 0.2908 0.9618N/A 0.082418 0.188961 0.363341 Hepatocellular mRNA (1.0000) (1.0000)(1.0000) Carcinoma Expression, (TCGA, RSEM (Batch PanCancer normalizedfrom Atlas) Illumina HiSeq_RNASeq V2)

Example 7: Expression Analysis of Nectin-4 in 6 Cell Lines

1. Study Objective

The objective of the study was to evaluate the expression of Nectin-4 in6 cell lines by flow cytometry, including 2 Breast cancer (T-47D,MDA-MB-468), 3 Lung cancer (NCI-H292, NCI-H322, NCI-H526) and 1fibrosarcoma (HT-1080) cell lines).

2. Panel Design

Panel for FCM in T-47D, MDA-MB-468, NCI-H292, NCI-H322 and HT-1080

Fluorochrome Blank Isotype Panel PE — Isotype ctrl Nectin-4

Panel for NCI-H526

Fluorochrome Blank Isotype Panel PE — Isotype ctrl Nectin-4 BV421Live/Dead Live/Dead Live/Dead3. Material3.1. SampleCell Lines List

Cancer Culture Item Cell lines Type Vendor Properties Culture Media 1T-47D Breast ATCC- adherent RPMI-1640 + 0.2 cancer HTB-133 Units/mlbovine insulin + 10% FBS 2 MDA-MB- Breast ATCC- adherent Leibovitz's L-468 cancer HTB-132 15 + 10% FBS 3 NCI-H292 Lung 91091815 adherentRPMI-1640 + 10% FBS 4 NCI-H322 Lung 95111734 adherent RPMI-1640 + 2 mMGlutamine + 10% FBS 5 NCI-H526 Lung CRL-5811 round RPMI-1640 + clustersin 10% FBS suspension 6 HT1080 Fibro- ECACC- adherent EMEM + 2 mMsarcoma 85111505 Glutamine + 1% Non Essential Amino Acids (NEAA) + 10%FBS3.2. ReagentsAntibodies and Kit for Flow Cytometry Analysis

Fluorescence Marker Catalog Provider Comment PE Nectin-4 FAB2659P R&DAAAO0217021 PE Isotype control IC0041P R&D From BICY- IgG2b 20161117ADPBS (Corning-21-031-CV)Staining buffer (eBioscience-00-4222)Fixation buffer (BD-554655)3.3. InstrumentsEppendorf Centrifuge 5810RBD FACS Canto Flow Cytometer (BD)4. Experimental Methods and Procedures4.1. Sample Collection

Harvest the cell lines growing in an exponential growth phase. Countcells by haemocytometer with Trypan blue staining. Centrifuge the cellsat 400×g for 5 min at 4° C., wash cells for two times with stainingbuffer, and suspend the cells in staining buffer to 1×10⁷/mL.

4.2. Antibody Staining

-   1) Aliquoted 100 μL cell suspension to each well of a 96-well    V-plate.-   2) Added Isotype control or antibodies into suspension cells and    incubated for 30 min at 4° C. in the dark.-   3) Washed cells 2× by centrifugation at 400×g for 5 min at 4° C. and    discarded supernatant.-   4) Resuspended cells with 100 μL fixation buffer and incubated for    30 min at 4° C. in the dark.-   5) Washed cells 2× by centrifugation at 300×g for 5 min at 4° C. and    removed supernatant-   6) Resuspended cells in 400 μL staining buffer.-   7) Analyzed the FACS data using FlowJo V10 software.    4.3. Data Analysis

All the FACS data was analyzed by Flowjo V10 software and Graphpad Prismor Excel software.

5. Results

5.1 Gate Strategy for Panel

Gating strategy for Nectin-4 is shown in FIGS. 8-11.

5.2. Data Analysis

5.2.1. Viability of Cell Lines

The viability of cell lines was as below.

Viable cells/ No. Cell line Cancer Type Viability million 1 T-47D Breast98.1 8.6 2 MDA-MB-468 Breast 98.7 5.3 3 NCI-H292 Lung 98.7 10.4 4NCI-H322 Lung 98.5 6.6 5 NCI-H526 Lung 79.9 4.0 6 HT1080 Fibrosarcoma98.0 14.75.2.2. The Positive Expression of Nectin-4 in Cell Lines

Positive expression and MFI of Nectin-4 in 6 cell lines were as list.

No. Cell line Nectin-4 MFI-Isotype MFI-Panel 1 T-47D 99.0% 132 1808 2MDA-MB-468 99.0% 184 2324 3 NCI-H292 97.9% 180 729 4 NCI-H322 99.1% 1451655 5 NCI-H526 0.21% 104 91.3 6 HT1080 1.53% 134 1346. Discussion

There was a high expression of Nectin-4 in Breast cancer T-47D (99.0%),MDA-MB-468 (99.0%) and lung cancer NCI-H292 (97.9%), NCI-H322 (99.1%).In NCI-H526 and HT-1080, no expression of Nectin-4 was found.

Example 8: Expression Analysis of Nectin-4 in 9 CDX Cell Lines by FlowCytometry

1. Study Objective

The objective of this project is to evaluate the surface expression ofNectin-4 (PVRL-4) in 9 cell lines, including 1 Breast cancer(MDA-MB-468), 4 Lung cancer (NCI-H292, NCI-H358, NCI-H526, A549), 1Pancreatic cancer (Panc02.13), 2 Colorectal cancer (HCT-116, HT-29) and1 Bladder cancer (HT1376) cell lines.

2. Panel Design

Panel for FCM in 9 Cell Lines

Fluorochrome Blank Isotype Panel PE — Isotype control IgG2b Nectin-4BV421 Live/Dead Live/Dead Live/Dead3. Materials3.1 SamplesCell Lines List

Cancer Culture Item Cell Line Type Vendor Properties Culture Media 1HT1376 Bladder ATCC- adherent EMEM + 10% CRL-1472 FBS 2 MDA-MB- BreastATCC- adherent Leibovitz's 468 HTB-132 L-15 + 10% FBS 3 HCT-116 Colo-ATCC- adherent RPMI 1640 + rectal CCL-247 10% FBS 4 HT-29 Colo- ATCC-adherent McCoy's 5a + rectal HTB-38 10% FBS 5 A549 Lung ATCC- adherentF-12K + 10% CCL-185 FBS 6 NCI-H292 Lung ECACC- suspension RPMI 1640 +91091815 10% FBS 7 NCI-H358 Lung ECACC- adherent RPMI 1640 + 9511173310% FBS 8 NCI-526 Lung ATCC- adherent RPMI 1640 + CRL-5811 10% FBS 9Panc02.13 Pan- ATCC- adherent RPMI-1640 + creas CRL-2554 15% FBS + 5ug/ml human insulin3.2. Reagents

-   -   1) DPBS (Corning, 21-031-CV)    -   2) Trypsin 0.25% (Invitrogen-25200072)    -   3) Staining buffer (eBioscience, 00-4222)    -   4) Fixation buffer (BD, 554655)    -   5) Antibody

Fluorescence Marker Catalog Vendor Comment PE Nectin-4 FAB2659P R&DAAAO0217021 PE Mouse IgG2b IC0041P R&D BV421 Live/Dead L34964 Invitrogen—3.3. InstrumentsEppendorf Centrifuge 5810RBD FACS Canto Flow Cytometer (BD)4. Experimental Methods and Procedures4.1 Cell CultureCell Thawing

-   1) Cleaned the frozen vials with 70% alcohol and quickly thawed    vials in 37° C. water bath.-   2) Centrifuged cell suspension at approximately 1000 rpm for 5    minutes, removed the supernatant and added pre-warming medium into    the flasks.-   3) Incubated culture flasks at 37° C., 5% CO₂ incubator.    Cell Passage-   1) Warmed medium and trypsin in 37° C. water bath.-   2) Removed culture medium and rinsed the cell layer with DPBS.-   3) Added 5 mL 0.25% trypsin solution to flask and diluted trypsin    with 5 mL medium.-   4) Centrifuged cell suspension at 1000 rpm for 5 min.-   5) Added 15 mL fresh medium and re-suspended cells by pipetting    gently.-   6) Added appropriate cell suspension to new culture flasks.-   7) Incubated culture flasks at 37° C., 5% CO₂ incubator.    4.2. Samples Collection

Harvested the cell lines growing in an exponential growth phase. Countedcells with Trypan blue staining. Centrifuged the cells at 400×g for 5min at 4° C., washed cells with staining buffer for twice, and suspendedthe cells in staining buffer to 5×10⁶/mL.

4.3. Antibody Staining

Aliquoted 100 μL cell suspension to each well of a 96-well V-plate.Added Isotype control or antibodies into suspension cells and incubatedfor 30 min at 4° C. in the dark. Washed cells 2 times by centrifugationat 400×g for 5 min at 4° C. and discarded supernatant. Re-suspendedcells in 300 μL staining buffer. Analyzed the FACS data using Flow JoV10 software.

4.4. Data Analysis

All the FACS data was analyzed by Flow Jo V10 software and GraphPadPrism or Excel software.

5. Results

5.1. Gate Strategy for Panel

Gating strategy for Nectin-4 is shown in FIGS. 12-16.

5.2. Data Analysis

Positive expression and MFI of Nectin-4 in 9 cell lines were as list.

No. Cell Line Nectin-4 MFI-Isotype MFI-Panel 1 HT1376 92.4% 36.2 803 2MDA-MB-468 97.1% 28.9 460 3 HCT-116 1.85% 14.5 15.6 4 HT-29 40.0% 20.588.3 5 A549 1.07% 20.5 21.6 6 NCI-H292 71.1% 22.9 149 7 NCI-H358 90.1%26.5 361 8 NCI-526 1.22% 8.33 12.1 9 Panc02.13 51.9% 36.2 1286. Discussion

There was a high expression of Nectin-4 in Bladder cancer HT-1376(92.4%), Breast cancer MDA-MB-468 (97.1%) and lung cancer NCI-H358(90.1%). A medium expression of Nectin-4 was found in HT-29 (40.0%),NCI-H292 (71.1%) and Panc02.13 (51.9%). In HCT-116, A549 and NCI-526, noexpression of Nectin-4 was found. This data will be used to guide modelselection for efficacy studies.

Example 9: In Vivo Efficacy Studies Example 9.1: In Vivo Efficacy Studyof Test Articles in Treatment of A549 Xenograft in Balb/c Nude Mice

1. Study Objective

The objective of the research is to evaluate the in vivo anti-tumorefficacy of test articles in treatment of A549 xenograft in Balb/c nudemice.

2. Experimental Design

Dosing Dose Volume Dosing Group Treatment n (mg/kg) (μl/g) RouteSchedule 1 Vehicle 5 — 10 iv qw 2 BCY8245 3 3 mg/kg 10 iv qw 3 BCY8245 33 mg/kg 10 iv biw 4 BCY8245 3 5 mg/kg 10 iv qw3. MaterialsAnimals and Housing Condition3.1.1. AnimalsSpecies: Mus MusculusStrain: Balb/c nudeAge: 6-8 weeksSex: femaleBody weight: 18-22 gNumber of animals: 41 mice plus spare3.1.2. Housing Condition

The mice were kept in individual ventilation cages at constanttemperature and humidity with 3 or 5 animals in each cage.

-   -   Temperature: 20˜26° C.    -   Humidity 40-70%.        Cages: Made of polycarbonate. The size is 300 mm×180 mm×150 mm.        The bedding material is corn cob, which is changed twice per        week.        Diet: Animals had free access to irradiation sterilized dry        granule food during the entire study period.        Water: Animals had free access to sterile drinking water.        Cage identification: The identification labels for each cage        contained the following information: number of animals, sex,        strain, the date received, treatment, study number, group number        and the starting date of the treatment.        Animal identification: Animals were marked by ear coding.        4. Experimental Methods and Procedures        4.1. Cell Culture

The A549 tumor cells were maintained in vitro as a monolayer culture inF-12K medium supplemented with 10% heat inactivated fetal bovine serumat 37° C. in an atmosphere of 5% CO₂ in air. The tumor cells wereroutinely subcultured twice weekly by trypsin-EDTA treatment. The cellsgrowing in an exponential growth phase were harvested and counted fortumor inoculation.

4.2. Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank with A549tumor cells (5×10⁶) in 0.2 ml of PBS for tumor development. 41 animalswere randomized when the average tumor volume reached 158 mm³. The testarticle administration and the animal numbers in each group were shownin the experimental design table.

4.3. Testing Article Formulation Preparation

Test Con. article (mg/ml) Formulation Vehicle — 25 mM Histidine pH 7 10%sucrose BCY8245 0.5 Dilute 400 μl 1 mg/ml BCY8245 stock with 400 μlHistidine buffer BCY8245 0.3 Dilute 240 μl 1 mg/ml BCY8245 stock with560 μl Histidine buffer4.4. Sample Collection

At the end of study, the tumor of all groups were collected at 2 h postlast dosing.

5. Results

5.1. Tumor Growth Curves

The tumor growth curve is shown in FIG. 17.

5.2. Tumor Volume Trace

Mean tumor volume over time in female Balb/c nude mice bearing A549xenograft is shown in Table 21.

TABLE 21 Tumor volume trace over time Days after the start of treatmentGr. Treatment 0 2 5 7 9 12 14 1 Vehicle, 158 ± 13 235 ± 24 278 ± 26 346± 39 387 ± 35 471 ± 45 568 ± 49 qw 2 BCY8245, 157 ± 10 208 ± 15 197 ± 25257 ± 40 293 ± 41 341 ± 54 356 ± 53 3 mpk, qw 3 BCY8245, 157 ± 14 183 ±27 158 ± 16 184 ± 6  182 ± 8  190 ± 15 194 ± 27 3 mpk, biw 4 BCY8245,157 ± 14 179 ± 22 147 ± 10 172 ± 23 173 ± 24 204 ± 36 228 ± 33 5 mpk, qw5.3. Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for test articles in the A549 xenograftmodel was calculated based on tumor volume measurements at day 14 afterthe start of treatment.

TABLE 22 Tumor growth inhibition analysis Tumor P value Volume T/C^(b)TGI compared Gr Treatment (mm³)^(a) (%) (%) with vehicle 1 Vehicle, qw568 ± 49 — — — 2 BCY8245, 356 ± 53 62.8 51.4 p < 0.05  3 mpk, qw 3BCY8245, 194 ± 27 34.2 90.8 p < 0.001 3 mpk, biw 4 BCY8245, 228 ± 3340.2 82.6 p < 0.001 5 mpk, qw ^(a)Mean ± SEM. ^(b)Tumor GrowthInhibition is calculated by dividing the group average tumor volume forthe treated group by the group average tumor volume for the controlgroup (T/C).6. Results Summary and Discussion

In this study, the therapeutic efficacy of test articles in the A549xenograft model was evaluated. The measured tumor volumes of alltreatment groups at various time points are shown in FIG. 17 and Tables21 and 22.

The mean tumor size of vehicle treated mice reached 568 mm³ on day 14.BCY8245 at 3 mg/kg, qw (TV=356 mm³, TGI=51.4%, p<0.05), 3 mg/kg, biw(TV=194 mm³, TGI=90.8%, p<0.01) and 5 mg/kg, qw (TV=228 mm³, TGI=82.6%,p<0.001) produced significant anti-tumor antitumor activity in dose ordose-frequency dependent manner.

Animals in BCY8245 groups maintained the bodyweight well. In this cellline, which shows minimal expression of Nectin-4 in FACS studies, tumorgrowth is restrained by BCY8245 but the tumor does not undergoregression, emphasising the target driven requirement for optimalefficacy.

Example 9.2: In Vivo Efficacy Study of Test Articles in Treatment ofHCT116 Xenograft in Balb/c Nude Mice

1. Study Objective

The objective of the research is to evaluate the in vivo anti-tumorefficacy of test articles in treatment of HCT116 xenograft in Balb/cnude mice.

2. Experimental Design

Dose Dosing Dosing Group Treatment n (mg/kg) Volume (μl/g) RouteSchedule 1 Vehicle 5 — 10 iv qw 2 BCY8245 3 3 10 iv qw 3 BCY8245 3 3 10iv biw 4 BCY8245 3 5 10 iv qw3. Materials3.1. Animals and Housing Condition3.1.1. AnimalsSpecies: Mus MusculusStrain: Balb/c nudeAge: 6-8 weeksSex: femaleBody weight: 18-22 gNumber of animals: 41 mice plus spare3.1.2. Housing Condition

The mice were kept in individual ventilation cages at constanttemperature and humidity with 3 or 5 animals in each cage.

-   -   Temperature: 20˜26° C.    -   Humidity 40-70%.        Cages: Made of polycarbonate. The size is 300 mm×180 mm×150 mm.        The bedding material is corn cob, which is changed twice per        week.        Diet: Animals had free access to irradiation sterilized dry        granule food during the entire study period.        Water: Animals had free access to sterile drinking water.        Cage identification: The identification labels for each cage        contained the following information: number of animals, sex,        strain, the date received, treatment, study number, group number        and the starting date of the treatment.        Animal identification: Animals were marked by ear coding.        4. Experimental Methods and Procedures        4.1 Cell Culture

The HCT116 cells were maintained in medium supplemented with 10% heatinactivated fetal bovine serum at 37° C. in an atmosphere of 5% CO₂ inair. The tumor cells were routinely subcultured twice weekly. The cellsgrowing in an exponential growth phase were harvested and counted fortumor inoculation.

4.2. Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank with HCT116tumor cells (5.0×10⁶) in 0.2 ml of PBS for tumor development. 41 animalswere randomized when the average tumor volume reached 166 mm³. The testarticle administration and the animal numbers in each group were shownin the experimental design table.

4.3. Testing Article Formulation Preparation

Test Con. article (mg/ml) Formulation Vehicle — 25 mM Histidine pH 7 10%sucrose BCY8245 0.5 Dilute 400 μl 1 mg/ml BCY8245 stock with 400 μlHistidine buffer BCY8245 0.3 Dilute 240 μl 1 mg/ml BCY8245 stock with560 μl Histidine buffer4.4. Sample Collection

At the end of study on day 14, tumors from group 1 and 2 were collectedfor FFPE. For group 4, plasma were collected at 5 min, 15 min, 30 min,60 min and 120 min post dosing. Tumors were also collected and stored at−80° C.

5. Results

5.1. Tumor Growth Curves

The tumor growth curve is shown in FIG. 18.

5.2. Tumor Volume Trace

Mean tumor volume over time in female Balb/c nude mice bearing HCT116xenograft is shown in Table 23.

TABLE 23 Tumor volume trace over time Days after the start of treatmentGroup Treatment 0 2 4 7 9 12 14 1 Vehicle, 166 ± 12 219 ± 21 323 ± 29397 ± 28 488 ± 36 630 ± 49 769 ± 71 qw 2 BCY8245, 167 ± 11 209 ± 13 227± 17 269 ± 33 324 ± 39 348 ± 27 425 ± 28 3 mpk, qw 3 BCY8245, 166 ± 18229 ± 40 215 ± 42 213 ± 49 213 ± 48 206 ± 55 197 ± 50 3 mpk, biw 4BCY8245, 166 ± 35 201 ± 42 176 ± 15 183 ± 17 170 ± 16 125 ± 18 134 ± 125 mpk, qw5.3. Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for test articles in the HCT116 xenograftmodel was calculated based on tumor volume measurements at day 14 afterthe start of the treatment.

TABLE 24 Tumor growth inhibition analysis Tumor Volume T/C^(b) TGI GroupTreatment (mm³)^(a) (%) (%) P value 1 Vehicle, qw 769 ± 71 — — — 2BCY8245, 425 ± 28 55.2 57.1 p < 0.001 3 mpk, qw 3 BCY8245, 197 ± 50 25.694.9 p < 0.001 3 mpk, biw 4 BCY8245, 134 ± 12 17.4 105.2 p < 0.001 5mpk, qw ^(a)Mean ± SEM; ^(b)Tumor Growth Inhibition is calculated bydividing the group average tumor volume for the treated group by thegroup average tumor volume for the control group (T/C).6. Results Summary and Discussion

In this study, the therapeutic efficacy of test articles in the HCT116xenograft model was evaluated. The measured tumor volumes of alltreatment groups at various time points are shown in FIG. 18 and Tables23 and 24.

The mean tumor size of vehicle treated mice reached 769 mm³ on day 14after the start of treatment. BCY8245 at 3 mg/kg, qw (TV=425 mm³,TGI=57.1%, p<0.001), 3 mg/kg, biw (TV=197 mm³, TGI=94.9%, p<0.001) and 5mg/kg, qw (TV=134 mm³, TGI=105.2%, p<0.001) produced significantanti-tumor antitumor activity in dose or dose-frequency dependentmanner.

In this study, animals in all of 5 mg/kg qw groups lost over average 10%bodyweight.

In this cell line, which shows minimal expression of Nectin-4 in FACSstudies, tumor growth is restrained by BCY8245 but the tumor does notundergo regression, emphasising the target driven requirement foroptimal efficacy.

Example 9.3: In Vivo Efficacy Study of Test Articles in Treatment ofHT-1376 Xenograft in CB17-SCID Mice

1. Study Objective

The objective of the research is to evaluate the in vivo anti-tumorefficacy of test articles in treatment of HT-1376 xenograft in CB17-SCIDmice.

2. Experimental Design

Dosing Dose Volume Dosing Group Treatment n (mg/kg) (μl/g) RouteSchedule 1 Vehicle 5 — 10 iv qw 2 BCY8245 3 3 mg/kg 10 iv qw 3 BCY8245 33 mg/kg 10 iv biw 4 BCY8245 3 5 mg/kg 10 iv qw3. Materials3.1 Animals and Housing Condition3.1.1. AnimalsSpecies: Mus MusculusStrain: CB17-SCIDAge: 6-8 weeksSex: femaleBody weight: 18-22 gNumber of animals: 41 mice plus spare3.1.2. Housing Condition

The mice were kept in individual ventilation cages at constanttemperature and humidity with 3 or 5 animals in each cage.

-   -   Temperature: 20˜26° C.    -   Humidity 40-70%.        Cages: Made of polycarbonate. The size is 300 mm×180 mm×150 mm.        The bedding material is corn cob, which is changed twice per        week.        Diet: Animals had free access to irradiation sterilized dry        granule food during the entire study period.        Water: Animals had free access to sterile drinking water.        Cage identification: The identification labels for each cage        contained the following information: number of animals, sex,        strain, the date received, treatment, study number, group number        and the starting date of the treatment.        Animal identification: Animals were marked by ear coding.        4. Experimental Methods and Procedures        4.1 Cell Culture

The HT-1376 tumor cells will be maintained in EMEM medium supplementedwith 10% heat inactivated fetal bovine serum at 37° C. in an atmosphereof 5% CO₂ in air. The tumor cells will be routinely subcultured twiceweekly. The cells growing in an exponential growth phase will beharvested and counted for tumor inoculation.

4.2 Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank with HT-1376tumor cells (5×10⁶) with Matrigel (1:1) in 0.2 ml of PBS for tumordevelopment. 41 animals were randomized when the average tumor volumereached 153 mm³. The test article administration and the animal numbersin each group were shown in the experimental design table.

4.3. Testing Article Formulation Preparation

Test Con. article (mg/ml) Formulation Vehicle — 25 mM Histidine pH 7 10%sucrose BCY8245 0.5 Dilute 400 μl 1 mg/ml BCY8245 stock with 400 μlHistidine buffer BCY8245 0.3 Dilute 240 μl 1 mg/ml BCY8245 stock with560 μl Histidine buffer4.4. Sample Collection

At the end of study, the plasma of group 4 was collected at 5 min, 15min, 30 min, 60 min and 120 min post last dosing. The tumor of group 4was collected at 2 h post last dosing. The tumor of groups 1, 2 and 3were collected at 2 h post last dosing.

5. Results

5.1. Tumor Growth Curves

The tumor growth curve is shown in FIG. 19.

5.2. Tumor Volume Trace

Mean tumor volume over time in female CB17-SCID mice bearing HT-1376xenograft is shown in Table 25.

TABLE 25 Tumor volume trace over time Days after the start of treatmentGr. Treatment 0 2 5 7 9 12 14 1 Vehicle, qw 153 ± 16 266 ± 30 398 ± 41529 ± 56 721 ± 76 908 ± 91 1069 ± 90  2 BCY8245, 153 ± 26 254 ± 53 298 ±69 398 ± 61 468 ± 73 502 ± 67 603 ± 76 3 mpk, qw 3 BCY8245, 154 ± 30 248± 58 203 ± 15 273 ± 45 356 ± 50 391 ± 53 407 ± 53 3 mpk, biw 4 BCY8245,153 ± 15 237 ± 41 228 ± 36 317 ± 31 394 ± 20 438 ± 31 465 ± 33 5 mpk, qw5.3. Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for Test articles in the HT-1376 xenograftmodel was calculated based on tumor volume measurements at day 14 afterthe start of treatment.

TABLE 26 Tumor growth inhibition analysis Tumor P value Volume T/C^(b)TGI compared Gr Treatment (mm³)^(a) (%) (%) with vehicle 1 Vehicle, qw1069 ± 90  — — — 2 BCY8245, 603 ± 76 56.4 50.9 p < 0.01  3 mpk, qw 3BCY8245, 407 ± 53 38.1 72.3 p < 0.001 3 mpk, biw 4 BCY8245, 465 ± 3343.5 66.0 p < 0.001 5 mpk, qw ^(a)Mean ± SEM. ^(b)Tumor GrowthInhibition is calculated by dividing the group average tumor volume forthe treated group by the group average tumor volume for the controlgroup (T/C).6. Results Summary and Discussion

In this study, the therapeutic efficacy of test articles in the HT-1376xenograft model was evaluated. The measured tumor volumes of alltreatment groups at various time points are shown in FIG. 19 and Tables25 and 26.

The mean tumor size of vehicle treated mice reached 1069 mm³ on day 14.BCY8245 at 3 mg/kg, qw (TV=603 mm³, TGI=50.9%, p<0.01), 3 mg/kg, biw(TV=407 mm³, TGI=72.3%, p<0.001) and 5 mg/kg, qw (TV=465 mm³, TGI=66.0%,p<0.001) produced significant antitumor activity. In this study, BCY8245at 5 mg/kg qw caused over 10% animal bodyweight loss during thetreatment schedule.

Example 9.4: In Vivo Efficacy Study of Test Articles in Treatment ofMDA-MB-468 Xenograft in Balb/c Nude Mice

1. Study Objective

The objective of the research is to evaluate the in vivo anti-tumorefficacy of test articles in treatment of MDA-MB-468 xenograft in Balb/cnude mice.

2. Experimental Design

Dosing Dose Volume Dosing Group Treatment n (mg/kg) (μl/g) RouteSchedule 1 Vehicle 5 — 10 iv qw 2 BCY8245 3 3 mg/kg 10 iv qw 3 BCY8245 33 mg/kg 10 iv biw 4 BCY8245 3 5 mg/kg 10 iv qw3. Materials3.1. Animals and Housing Condition3.1.1. AnimalsSpecies: Mus MusculusStrain: Balb/c nudeAge: 6-8 weeksSex: femaleBody weight: 18-22 gNumber of animals: 41 mice plus spare3.1.2. Housing Condition

The mice were kept in individual ventilation cages at constanttemperature and humidity with 3 or 5 animals in each cage.

-   -   Temperature: 20˜26° C.    -   Humidity 40-70%.        Cages: Made of polycarbonate. The size is 300 mm×180 mm×150 mm.        The bedding material is corn cob, which is changed twice per        week.        Diet: Animals had free access to irradiation sterilized dry        granule food during the entire study period.        Water: Animals had free access to sterile drinking water.        Cage identification: The identification labels for each cage        contained the following information: number of animals, sex,        strain, the date received, treatment, study number, group number        and the starting date of the treatment.        Animal identification: Animals were marked by ear coding.        4. Experimental Methods and Procedures        4.1. Cell Culture

The tumor cells were maintained in Leibovitz's L-15 medium supplementedwith 10% heat inactivated fetal bovine serum at 37° C. in an atmosphereof 5% CO₂ in air. The tumor cells were routinely subcultured twiceweekly. The cells growing in an exponential growth phase were harvestedand counted for tumor inoculation.

4.2. Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank withMDA-MB-468 tumor cells (10×10⁶) in 0.2 ml of PBS supplemented with 50%matrigel for tumor development. 41 animals were randomized when theaverage tumor volume reached 196 mm³. The test article administrationand the animal numbers in each group were shown in the experimentaldesign table.

4.3. Testing Article Formulation Preparation

Test Con. article (mg/ml) Formulation Vehicle — 25 mM Histidine pH 7 10%sucrose BCY8245 1 Dissolve 10.56 mg BCY8245 in 10.518 ml Histidinebuffer BCY8245 0.5 Dilute 400 μl 1 mg/ml BCY8245 stock with 400 μlHistidine buffer BCY8245 0.3 Dilute 240 μl 1 mg/ml BCY8245 stock with560 μl Histidine buffer4.4. Sample Collection

At the day 21 of study, the plasma of group 2 was collected at 5 min, 15min, 30 min, 60 min and 120 min post last dosing. The tumors of groups 1and 3 were collected at 2 h post last dosing. The animals in group 4were kept running for another 21 days without any dosing, and the tumorsof these groups were collected on day 42.

5. Results

5.1. Tumor Growth Curves

The tumor growth curve is shown in FIG. 20.

5.2. Tumor Volume Trace

Mean tumor volume over time in female Balb/c nude mice bearingMDA-MB-468 xenograft is shown in Table 27 and 28.

5.3. Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for test articles in the MDA-MB-468xenograft model was calculated based on tumor volume measurements at day21 after the start of the treatment.

TABLE 27 Tumor volume trace over time (Day 0 to day 21) Days after thestart of treatment Gr. Treatment 0 2 4 7 9 11 14 16 18 21 1 Vehicle, qw199 ± 6  217 ± 9  235 ± 15 274 ± 14 291 ± 14 314 ± 20 348 ± 24 374 ± 33398 ± 39 447 ± 39 2 BCY8245, 194 ± 12 192 ± 26 184 ± 20 131 ± 20 113 ±17 104 ± 13  94 ± 25  81 ± 23  87 ± 23  85 ± 31 3 mpk, qw 3 BCY8245, 195± 33 193 ± 27 154 ± 20 103 ± 20  83 ± 16  67 ± 14  49 ± 11  45 ± 14  32± 12 22 ± 4 3 mpk, biw 4 BCY8245, 199 ± 28 193 ± 11 135 ± 4  98 ± 5 58 ±5 49 ± 5 47 ± 2 37 ± 4 35 ± 3 29 ± 3 5 mpk, qw

TABLE 28 Tumor volume trace over time (Day 23 to day 42) Days after thestart of treatment Gr. Treatment 23 25 28 32 35 39 42 4 BCY8245, 35 ± 548 ± 5 37 ± 7 28 ± 6 24 ± 4 28 ± 6 26 ± 6 5 mpk, qw

TABLE 29 Tumor growth inhibition analysis Tumor T/C^(b) TGI P value GrTreatment Volume (%) (%) with 1 Vehicle, qw 447 ± 39 — — — 2 BCY8245, 85 ± 31 18.9 144.2 p < 0.001 3 mpk, qw 3 BCY8245, 22 ± 4 4.9 169.8 p <0.001 3 mpk, biw 4 BCY8245, 29 ± 3 6.6 168.4 p < 0.001 5 mpk, qw^(a)Mean ± SEM. ^(b)Tumor Growth Inhibition is calculated by dividingthe group average tumor volume for the treated group by the groupaverage tumor volume for the control group (T/C).6. Results Summary and Discussion

In this study, the therapeutic efficacy of test articles in theMDA-MB-468 xenograft model was evaluated. The measured tumor volumes ofall treatment groups at various time points are shown in FIG. 20 andTables 27 to 29.

The mean tumor size of vehicle treated mice reached 447 mm³ on day 21.BCY8245 at 3 mg/kg, qw (TV=85 mm³, TGI=144.2%, p<0.001), 3 mg/kg, biw(TV=22 mm³, TGI=169.8%, p<0.001) and 5 mg/kg, qw (TV=29 mm³, TGI=168.4%,p<0.001) produced significant anti-tumor antitumor activity in dose ordose-frequency dependent manner.

The dosing of 5 mg/kg groups were suspended from day 21, the tumorsdidn't show any relapse during extra 3 weeks' monitoring schedule. Inthis cell line, which shows high expression of Nectin-4 in FACS studies,BCY8245 causes regression of the tumor emphasizing the target drivennature of optimal efficacy.

Example 9.5: In Vivo Efficacy Study of Test Articles in Treatment ofNCI-H292 Xenograft in Balb/c Nude Mice

1. Study Objective

The objective of the research is to evaluate the in vivo anti-tumorefficacy of test articles in treatment of NCI-H292 xenograft in Balb/cnude mice.

2. Experimental Design

Dosing Dose Volume Dosing Group Treatment n (mg/kg) (μl/g) RouteSchedule 1 Vehicle 5 — 10 iv qw 2 BCY8245 3 3 mg/kg 10 iv qw 3 BCY8245 33 mg/kg 10 iv biw 4 BCY8245 3 5 mg/kg 10 iv qw3. Materials3.1. Animals and Housing Condition3.1.1. AnimalsSpecies: Mus MusculusStrain: Balb/c nudeAge: 6-8 weeksSex: femaleBody weight: 18-22 gNumber of animals: 41 mice plus spare3.1.2. Housing Condition

The mice were kept in individual ventilation cages at constanttemperature and humidity with 3 or 5 animals in each cage.

-   -   Temperature: 20˜26° C.    -   Humidity 40-70%.        Cages: Made of polycarbonate. The size is 300 mm×180 mm×150 mm.        The bedding material is corn cob, which is changed twice per        week.        Diet: Animals had free access to irradiation sterilized dry        granule food during the entire study period.        Water: Animals had free access to sterile drinking water.        Cage identification: The identification labels for each cage        contained the following information: number of animals, sex,        strain, the date received, treatment, study number, group number        and the starting date of the treatment.        Animal identification: Animals were marked by ear coding.        4. Experimental Methods and Procedures        4.1. Cell Culture

The NCI-H292 tumor cells were maintained in vitro as a monolayer culturein RPMI-1640 medium supplemented with 10% heat inactivated fetal bovineserum at 37° C. in an atmosphere of 5% CO₂ in air. The tumor cells wereroutinely subcultured twice weekly by trypsin-EDTA treatment. The cellsgrowing in an exponential growth phase were harvested and counted fortumor inoculation.

4.2. Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank withNCI-H292 tumor cells (10×10⁶) in 0.2 ml of PBS for tumor development. 41animals were randomized when the average tumor volume reached 162 mm³.The test article administration and the animal numbers in each groupwere shown in the experimental design table.

4.3. Testing Article Formulation Preparation

Test Con. article (mg/ml) Formulation Vehicle — 25 mM Histidine pH 7 10%sucrose BCY8245 1 Dissolve 10.56 mg BCY8245 in 10.518 ml Histidinebuffer BCY8245 0.5 Dilute 400 μl 1 mg/ml BCY8245 stock with 400 μlHistidine buffer BCY8245 0.3 Dilute 240 μl 1 mg/ml BCY8245 stock with560 μl Histidine buffer4.4. Sample Collection

At the end of study, the tumor of all groups were collected at 2 h postlast dosing.

5. Results

5.1 Tumor Growth Curves

The tumor growth curve is shown in FIG. 21.

5.2. Tumor Volume Trace

Mean tumor volume over time in female Balb/c nude mice bearing NCI-H292xenograft is shown in Table 30.

TABLE 30 Tumor volume trace over time Days after the start of treatmentGr. Treatment 0 2 4 7 9 11 14 1 Vehicle, qw 161 ± 2 270 ± 14 357 ± 14448 ± 17 570 ± 16 720 ± 36 948 ± 61 2 BCY8245, 160 ± 5 220 ± 11 266 ± 15218 ± 23 167 ± 10 161 ± 36 149 ± 43 3 mpk, qw 3 BCY8245,  162 ± 13 243 ±19 211 ± 12 101 ± 11 100 ± 8  87 ± 7 65 ± 3 3 mpk, biw 4 BCY8245, 160 ±9 176 ± 7  191 ± 3  105 ± 8  82 ± 3  91 ± 14 83 ± 8 5 mpk, qw5.3. Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for test articles in the NCI-H292 xenograftmodel was calculated based on tumor volume measurements at day 14 afterthe start of treatment.

TABLE 31 Tumor growth inhibition analysis Tumor P value Volume T/C^(b)TGI compared Gr Treatment (mm³)^(a) (%) (%) with vehicle 1 Vehicle, qw948 ± 61 — — — 2 BCY8245, 149 ± 43 15.8 101.4 p < 0.001 3 mpk, qw 3BCY8245, 65 ± 3 6.9 112.2 p < 0.001 3 mpk, biw 4 BCY8245, 83 ± 8 8.8109.8 p < 0.001 5 mpk, qw ^(a)Mean ± SEM. ^(b)Tumor Growth Inhibition iscalculated by dividing the group average tumor volume for the treatedgroup by the group average tumor volume for the control group (T/C).6. Results Summary and Discussion

In this study, the therapeutic efficacy of test articles in the NCI-H292xenograft model was evaluated. The measured tumor volumes of alltreatment groups at various time points are shown in FIG. 21 and Tables30 and 31.

The mean tumor size of vehicle treated mice reached 948 mm³ on day 14.BCY8245 at 3 mg/kg, qw (TV=149 mm³, TGI=101.4%, p<0.001), 3 mg/kg, biw(TV=65 mm³, TGI=112.2%, p<0.001) and 5 mg/kg, qw (TV=83 mm³, TGI=109.8%,p<0.001) produced significant antitumor activity.

All of the test articles at 3 mg/kg, qw, 3 mg/kg, biw and 5 mg/kg, qwshowed comparable antitumor activity, the efficacy didn't furtherimprove when increasing the dosage or dose-frequency.

In this study, mice in all groups maintained the bodyweight well.

In this cell line, which shows high expression of Nectin-4 in FACSstudies, BCY8245 causes regression of the tumor emphasizing the targetdriven nature of optimal efficacy.

Example 9.6: In Vivo Efficacy Study of Test Articles in Treatment ofNCI-H526 Xenograft in Balb/c Nude Mice

1. Study Objective

The objective of the research is to evaluate the in vivo anti-tumorefficacy of test articles in treatment of NCI-H526 xenograft in Balb/cnude mice.

2. Experimental Design

Dosing Dose Volume Dosing Group Treatment n (mg/kg) (μl/g) RouteSchedule 1 Vehicle 3 — 10 iv qw 2 BCY8245 3 3 10 iv qw 3 BCY8245 3 3 10iv biw 4 BCY8245 3 5 10 iv qw3. Materials3.1. Animals and Housing Condition3.1.1. AnimalsSpecies: Mus MusculusStrain: Balb/c nudeAge: 6-8 weeksSex: femaleBody weight: 18-22 gNumber of animals: 21 mice plus spare3.1.2. Housing Condition

The mice were kept in individual ventilation cages at constanttemperature and humidity with 3 animals in each cage.

-   -   Temperature: 20˜26° C.    -   Humidity 40-70%.        Cages: Made of polycarbonate. The size is 300 mm×180 mm×150 mm.        The bedding material is corn cob, which is changed twice per        week.        Diet: Animals had free access to irradiation sterilized dry        granule food during the entire study period.        Water: Animals had free access to sterile drinking water.        Cage identification: The identification labels for each cage        contained the following information: number of animals, sex,        strain, the date received, treatment, study number, group number        and the starting date of the treatment.        Animal identification: Animals were marked by ear coding.        4. Experimental Methods and Procedures        4.1 Cell Culture

The NCI-H526 cells were maintained in medium supplemented with 10% heatinactivated fetal bovine serum at 37° C. in an atmosphere of 5% CO₂ inair. The tumor cells were routinely subcultured twice weekly. The cellsgrowing in an exponential growth phase were harvested and counted fortumor inoculation.

4.2. Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank withNCI-H526 tumor cells (5.0×10⁶) in 0.2 ml of PBS for tumor development.21 animals were randomized when the average tumor volume reached 181mm³. The test article administration and the animal numbers in eachgroup were shown in the experimental design table.

4.3. Testing Article Formulation Preparation

Test Con. article (mg/ml) Formulation Vehicle — 25 mM Histidine pH 7 10%sucrose BCY8245 0.5 Dilute 400 μl 1 mg/ml BCY8245 stock with 400 μlHistidine buffer BCY8245 0.3 Dilute 240 μl 1 mg/ml BCY8245 stock with560 μl Histidine buffer4.4. Sample Collection

At the end of study on day 14, all tumors were collected for FFPE.

5. Results

5.1. Tumor Growth Curves

The tumor growth curve is shown in FIG. 22.

5.2. Tumor Volume Trace

Mean tumor volume over time in female Balb/c nude mice bearing NCI-H526xenograft is shown in Table 32.

TABLE 32 Tumor volume trace over time Days after the start of treatmentGroup Treatment 0 2 4 7 9 12 14 1 Vehicle, qw 181 ± 32 262 ± 56 431 ± 90563 ± 72  729 ± 115 1076 ± 155 1365 ± 208 2 BCY8245, 180 ± 32 256 ± 51403 ± 72 545 ± 68  657 ± 83  1019 ± 155 1205 ± 79  3 mpk, qw 3 BCY8245,182 ± 43 232 ± 49 308 ± 79 440 ± 112 530 ± 121  810 ± 197 1109 ± 250 3mpk, biw 4 BCY8245, 180 ± 52 209 ± 66 236 ± 72 383 ± 119 375 ± 115 365 ±79  476 ± 103 5 mpk, qw5.3. Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for test articles in the NCI-H526 xenograftmodel was calculated based on tumor volume measurements at day 14 afterthe start of the treatment.

TABLE 33 Tumor growth inhibition analysis Tumor Volume T/C^(b) TGI GroupTreatment (mm³)^(a) (%) (%) P value 1 Vehicle, qw 1365 ± 208 — — — 2BCY8245, 1205 ± 79  88.3 13.4 p > 0.05 3 mpk, qw 3 BCY8245, 1109 ± 25081.3 21.6 p > 0.05 3 mpk, biw 4 BCY8245,  476 ± 103 34.9 75.0 p < 0.01 5mpk, qw ^(a)Mean ± SEM; ^(b)Tumor Growth Inhibition is calculated bydividing the group average tumor volume for the treated group by thegroup average tumor volume for the control group (T/C).6. Results Summary and Discussion

In this study, the therapeutic efficacy of test articles in the NCI-H526xenograft model was evaluated. The measured tumor volumes of alltreatment groups at various time points are shown in FIG. 22 and Tables32 and 33.

The mean tumor size of vehicle treated mice reached 1365³ on day 14after the start of treatment. BCY8245 at 3 mg/kg, qw (TV=1205 mm³,TGI=13.4%, p>0.05) and 3 mg/kg, biw (TV=1109 mm³, TGI=21.6%, p>0.05)showed slight antitumor activity, BCY8245 at 5 mg/kg, qw (TV=476 mm³,TGI=75.0%, p<0.01) showed significant antitumor activity. In this study,BCY8245 at 5 mg/kg biw caused over 10% animal bodyweight loss. In thiscell line, which shows minimal expression of Nectin-4 in FACS studies,tumor growth is restrained by BCY8245 but the tumor does not undergoregression, emphasising the target driven requirement for optimalefficacy.

Example 9.7: In Vivo Efficacy Study of Test Articles in Treatment ofPanc2.13 Xenograft in Balb/c Nude Mice

1. Study Objective

The objective of the research is to evaluate the in vivo anti-tumorefficacy of test articles in treatment of Panc2.13 xenograft in Balb/cnude mice.

2. Experimental Design

Dosing Dose Volume Dosing Group Treatment n (mg/kg) (μl/g) RouteSchedule 1 Vehicle 5 — 10 iv qw 2 BCY8245 3 3 mg/kg 10 iv qw 3 BCY8245 33 mg/kg 10 iv biw 4 BCY8245 3 5 mg/kg 10 iv qw3. Materials3.1. Animals and Housing Condition3.1.1. AnimalsSpecies: Mus MusculusStrain: Balb/c nudeAge: 6-8 weeksSex: femaleBody weight: 18-22 gNumber of animals: 41 mice plus spare3.1.2. Housing Condition

The mice were kept in individual ventilation cages at constanttemperature and humidity with 3 or 5 animals in each cage.

-   -   Temperature: 20˜26° C.    -   Humidity 40-70%.        Cages: Made of polycarbonate. The size is 300 mm×180 mm×150 mm.        The bedding material is corn cob, which is changed twice per        week.        Diet: Animals had free access to irradiation sterilized dry        granule food during the entire study period.        Water: Animals had free access to sterile drinking water.        Cage identification: The identification labels for each cage        contained the following information: number of animals, sex,        strain, the date received, treatment, study number, group number        and the starting date of the treatment.        Animal identification: Animals were marked by ear coding.        4. Experimental Methods and Procedures        4.1. Cell Culture

The Panc2.13 tumor cells will be maintained in RMPI1640 mediumsupplemented with 15% heat inactivated fetal bovine serum and 10units/ml human recombinant insulin at 37° C. in an atmosphere of 5% CO₂in air. The tumor cells will be routinely subcultured twice weekly. Thecells growing in an exponential growth phase will be harvested andcounted for tumor inoculation.

4.2. Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank withPanc2.13 tumor cells (5×10⁶) with Matrigel (1:1) in 0.2 ml of PBS fortumor development. 41 animals were randomized when the average tumorvolume reached 149 mm³. The test article administration and the animalnumbers in each group were shown in the experimental design table.

4.3. Testing Article Formulation Preparation

Test Con. article (mg/ml) Formulation Vehicle — 25 mM Histidine pH 7 10%sucrose BCY8245 0.5 Dilute 400 μl 1 mg/ml BCY8245 stock with 400 μlHistidine buffer BCY8245 0.3 Dilute 240 μl 1 mg/ml BCY8245 stock with560 μl Histidine buffer4.4. Sample Collection

At the end of study, the tumor of all groups were collected at 2 h postlast dosing.

5. Results

5.1. Tumor Growth Curves

The tumor growth curve is shown in FIG. 23.

5.2. Tumor Volume Trace

Mean tumor volume over time in female Balb/c nude mice bearing Panc2.13xenograft is shown in Table 34.

TABLE 34 Tumor volume trace over time Days after the start of treatmentGr. Treatment 0 2 4 7 9 11 14 1 Vehicle, qw 149 ± 12 202 ± 12 240 ± 9 321 ± 17 410 ± 27 479 ± 32 545 ± 17 2 BCY8245, 149 ± 34 160 ± 33 191 ±39 215 ± 53 242 ± 62 259 ± 59 271 ± 54 3 mpk, qw 3 BCY8245, 148 ± 46 170± 38 204 ± 57 216 ± 56 236 ± 59 241 ± 60 231 ± 57 3 mpk, biw 4 BCY8245,149 ± 18 180 ± 11 231 ± 33 242 ± 34 248 ± 40 231 ± 37 238 ± 40 5 mpk, qw5.3. Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for Test articles in the Panc2.13 xenograftmodel was calculated based on tumor volume measurements at day 14 afterthe start of treatment.

TABLE 35 Tumor growth inhibition analysis Tumor P value Volume T/C^(b)TGI compared Gr Treatment (mm³)^(a) (%) (%) with vehicle 1 Vehicle, qw545 ± 17 — — — 2 BCY8245, 271 ± 54 49.6 69.2 p < 0.01  3 mpk, qw 3BCY8245, 231 ± 57 42.3 79.1 p < 0.001 3 mpk, biw 4 BCY8245, 238 ± 4043.6 77.5 p < 0.001 5 mpk, qw ^(a)Mean ± SEM. ^(b)Tumor GrowthInhibition is calculated by dividing the group average tumor volume forthe treated group by the group average tumor volume for the controlgroup (T/C).6. Results Summary and Discussion

In this study, the therapeutic efficacy of test articles in the Panc2.13xenograft model was evaluated. The measured tumor volumes of alltreatment groups at various time points are shown in FIG. 23 and Tables34 and 35.

The mean tumor size of vehicle treated mice reached 545 mm³ on day 14.BCY8245 at 3 mg/kg, qw (TV=271 mm³, TGI=69.2%, p<0.01), 3 mg/kg, biw(TV=231 mm³, TGI=79.1%, p<0.001) and 5 mg/kg, qw (TV=238 mm³, TGI=77.5%,p<0.001) produced significant antitumor activity. In this study, animalsin all of 5 mg/kg qw groups lost over average 15% bodyweight. In thiscell line, which shows only moderate expression of Nectin-4 in FACSstudies, tumor growth is restrained by BCY8245 but the tumor does notundergo regression.

Example 9.8: In Vivo Efficacy Study of Test Articles in Treatment ofMDA-MB-468 Xenograft in Balb/c Nude Mice

1. Study Objective

The objective of the research is to evaluate the in vivo anti-tumorefficacy of BCY8245 and BCY8245 in combination with BCY8234 in treatmentof MDA-MB-468 xenograft in Balb/c nude mice to determine the role targetbinding has to play in optimal efficacy.

2. Experimental Design

Dose Dosing Group Treatment (mg/kg) N Route Schedule 1 Vehicle — 4 i.v.Qw, 3 weeks 2 BCY8245 0.3 4 i.v. Qw, 3 weeks 3 BCY8245 1 4 i.v. Qw, 3weeks 4 BCY8245 3 4 i.v. Qw, 3 weeks 5 BCY8245 + 1 + 300 4 i.v. Qw, 3weeks BCY8234 6 BCY8245 + 3 + 300 4 i.v. Qw, 3 weeks BCY8234 Note: N,the number of animals in each group.3. Materials3.1. Animals and Housing Condition3.1.1. AnimalsSpecies: Mus MusculusStrain: Balb/c nudeAge: 6-8 weeksSex: femaleBody weight: 18-22 gNumber of animals: 36 mice plus spare3.1.2. Housing Condition

The mice were kept in individual ventilation cages at constanttemperature and humidity with 4 animals in each cage.

-   -   Temperature: 20˜26° C.    -   Humidity 40-70%.        Cages: Made of polycarbonate. The size is 300 mm×180 mm×150 mm.        The bedding material is corn cob, which is changed twice per        week.        Diet: Animals had free access to irradiation sterilized dry        granule food during the entire study period.        Water: Animals had free access to sterile drinking water.        Cage identification: The identification labels for each cage        contained the following information: number of animals, sex,        strain, the date received, treatment, study number, group number        and the starting date of the treatment.        Animal identification: Animals were marked by ear coding.        4. Experimental Methods and Procedures        4.1. Cell Culture

The tumor cells were maintained in Leibovitz's L-15 medium supplementedwith 10% heat inactivated fetal bovine serum at 37° C. in an atmosphereof 0% CO₂ in air. The tumor cells were routinely subcultured twiceweekly. The cells growing in an exponential growth phase were harvestedand counted for tumor inoculation.

4.2. Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank withMDA-MB-468 tumor cells (10×10⁶) in 0.2 ml of PBS supplemented with 50%matrigel for tumor development. 36 animals were randomized when theaverage tumor volume reached 186 mm³. The test article administrationand the animal numbers in each group were shown in the experimentaldesign table.

4.3. Testing Article Formulation Preparation

Test Conc. article Purity (mg/ml) Formulation Vehicle — — 25 mMHistidine, pH 7 10% sucrose BCY8245 99.4% 1 Dissolve 5.0 mg BCY8245 in4.97 ml Histidine buffer¹ 0.03 Dilute 36 μl 1 mg/ml BCY8245 stock with1164 μl Histidine buffer 0.1 Dilute 120 μl 1 mg/ml BCY8245 stock with1080 μl Histidine buffer 0.3 Dilute 360 μl 1 mg/ml BCY8245 stock with840 μl Histidine buffer BCY8234 98.10% 30 Dissolve 147 mg BCY8234 in4.807 ml Histidine buffer4.4. Sample Collection

At the day 21 of study, the tumors of group 5 and 6 were collected forFFPE. At the end of the study, the tumors of group 3 was collected forFFPE.

5. Results

5.1. Tumor Growth Curve

The tumor growth curve is shown in FIG. 24.

5.2. Tumor Volume Trace

Mean tumor volume over time in female Balb/c nude mice bearingMDA-MB-468 xenograft is shown in Tables 36 to 38.

5.3. Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for test articles in the MDA-MB-468xenograft model was calculated based on tumor volume measurements at day21 after the start of the treatment.

6. Results Summary and Discussion

In this study, the therapeutic efficacy of test articles in theMDA-MB-468 xenograft model was evaluated. The measured tumor volumes ofall treatment groups at various time points are shown in FIG. 24 andTables 36 to 39.

The mean tumor size of vehicle treated mice reached 420 mm³ on day 21.BCY8245 at 1 mg/kg, qw (TV=204 mm³, TGI=92.1%, p<0.001), 3 mg/kg, qw(TV=27 mm³, TGI=164.9%, p<0.001) produced significant anti-tumoractivity in dose-dependent manner. BCY8245 at 0.3 mg/kg qw or biw didnot show any anti-tumor activity.

BCY8245 at 1 mg/kg, qw and 3 mg/kg, qw in combination with BCY8234 (thetoxin free cognate peptide) 300 mg/kg, qw produced significantanti-tumor activity (TV=242 mm³, TGI=75.4%, p<0.01) produced significantanti-tumor activity. When comparing with BCY8245 alone, the anti-tumoractivity of BCY8245 at 3 mg/kg was antagonized by BCY8234 at 300 mg/kg(p<0.001). This reduction in efficacy by the competing toxin-freepeptide demonstrates the importance of target binding for optimalefficacy. During the following monitoring schedule, the mice treatedwith BCY8245 1 mg/kg qw showed obvious tumor relapse, while the micetreated with BCY8245 3 mg/kg qw didn't show any tumor relapse.

TABLE 36 Tumor volume trace over time (Day 0 to day 21) Days after thestart of treatment Gr. Treatment 0 2 4 7 9 11 14 16 18 21 1 Vehicle, qw182 ± 15 196 ± 18 217 ± 18 260 ± 15 283 ± 19 302 ± 26 335 ± 27 362 ± 28386 ± 31 420 ± 37 2 BCY8245 188 ± 21 189 ± 19 211 ± 21 235 ± 28 252 ± 25253 ± 33 275 ± 26 277 ± 27 295 ± 26 300 ± 27 0.3 mpk, qw 3 BCY8245 185 ±21 187 ± 22 183 ± 22 190 ± 28 205 ± 29 195 ± 27 201 ± 25 197 ± 22 218 ±24 204 ± 19 1 mpk, qw 4 BCY8245 181 ± 14 171 ± 17 163 ± 10 141 ± 21 113± 16 92 ± 5 66 ± 4 58 ± 2 41 ± 2 27 ± 1 3 mpk, qw 5 BCY8245 + 184 ± 15189 ± 22 194 ± 28 212 ± 30 221 ± 34 221 ± 39 223 ± 36 211 ± 38 221 ± 51242 ± 67 BCY8234 1 + 300 mpk, qw 6 BCY8245 + 184 ± 16 178 ± 57 193 ± 36197 ± 46 179 ± 44 138 ± 41 137 ± 32 114 ± 24 110 ± 24  99 ± 18 BCY82343 + 300 mpk, qw

TABLE 37 Tumor volume trace over time (Day 23 to day 32) Days after thestart of treatment Gr. Treatment 23 25 28 30 32 1 Vehicle, qw 434 ± 35460 ± 38 504 ± 32 535 ± 46 548 ± 51 2 BCY8245 284 ± 14 268 ± 10 254 ± 15240 ± 21 241 ± 32 0.3 mpk, qw 3 BCY8245 200 ± 16 199 ± 13 210 ± 6  221 ±14 239 ± 16 1 mpk, qw 4 BCY8245 22 ± 3 19 ± 3 20 ± 3 15 ± 2 15 ± 1 3mpk, qw

TABLE 38 Tumor volume trace over time (Day 35 to day 91) Group 1,Vehicle, qw, dosed with Group 2, Group 3 Group 4, BCY8245 5 mpk BCY8245,BCY8245, BCY8245, Days on PG-D32 0.3 mpk, qw 1 mpk, qw 3 mpk, qw 35 455± 81 237 ± 33 255 ± 15  19 ± 2 37 373 ± 92 246 ± 40 292 ± 19  16 ± 2 39247 ± 48 248 ± 37 304 ± 36  14 ± 1 42 134 ± 18 245 ± 48 314 ± 42  14 ± 244 108 ± 3  251 ± 48 327 ± 42  18 ± 4 46 79 ± 3 248 ± 61 342 ± 55  19 ±4 49 63 ± 8 250 ± 65 356 ± 59  20 ± 4 51 62 ± 5 264 ± 68 374 ± 71  18 ±5 53  53 ± 12 268 ± 81 381 ± 87  22 ± 4 56  52 ± 13 271 ± 87 416 ± 10421 ± 4 58 58 ± 0 267 ± 95 433 ± 113 20 ± 5 60 61 ± 7  270 ± 108 464 ±119 18 ± 6 64  71 ± 23  276 ± 132 532 ± 154 23 ± 6 67  66 ± 14  269 ±137 550 ± 162 23 ± 5 71 73 ± 5  280 ± 155 565 ± 170 24 ± 7 74 82 ± 4 295 ± 167 594 ± 173 27 ± 8 78 90 ± 8  313 ± 194 612 ± 195 23 ± 6 81 104± 17  291 ± 192 639 ± 206 27 ± 8 84 110 ± 1   301 ± 194 695 ± 234 34 ± 788 106 ± 7   277 ± 194 743 ± 236 32 ± 7 91 110 ± 3   293 ± 209 771 ± 24026 ± 6

TABLE 39 Tumor growth inhibition analysis P value Combo Tumor comparedcompared Volume T/C^(b) TGI with With Gr Treatment (mm³)^(a) (%) (%)vehicle BCY8245 1 Vehicle, qw 420 ± 37 — — — — 2 BCY8245 300 ± 27 71.452.7 p > 0.05  0.3 mpk, qw 3 BCY8245 204 ± 19 48.6 92.1 p < 0.001 1 mpk,qw 4 BCY8245 27 ± 1 6.5 164.9 p < 0.001 3 mpk, qw 5 BCY8245 + 242 ± 6757.8 75.4 p < 0.01  p > 0.05  BCY8234 1 + 300 mpk, qw 6 BCY8245 +  99 ±18 23.5 135.9 p < 0.001 P < 0.001 BCY8234 3 + 300 mpk, qw ^(a)Mean ±SEM. ^(b)Tumor Growth Inhibition is calculated by dividing the groupaverage tumor volume for the treated group by the group average tumorvolume for the control group (T/C).

Example 9.9: In Vivo Efficacy Study of Test Articles in Treatment ofMDA-MB-468 Xenograft in Balb/c Nude Mice

1. Study Objective

The objective of the research is to evaluate the in vivo anti-tumorefficacy of BCY8245 alone or in combination with BCY8234 in treatment ofMDA-MB-468 xenograft in Balb/c nude mice.

2. Experimental Design

Dose Dosing Group Treatment (mg/kg) N Route Schedule 1 Vehicle — 5 i.v.Qw, 3 2 BCY8245 1 5 i.v. Qw, 3 3 BCY8245 3 5 i.v. Qw, 3 4 BCY8245 + 3 +300 = 300 5 i.v. Qw, 3 weeks BCY8234 Note: N, the number of animals ineach group.3. Materials3.1 Animals and Housing Condition3.1.1. AnimalsSpecies: Mus MusculusStrain: Balb/c nudeAge: 6-8 weeksSex: femaleBody weight: 18-22 gNumber of animals: 20 mice plus spare3.1.2. Housing Condition

The mice were kept in individual ventilation cages at constanttemperature and humidity with 5 animals in each cage.

-   -   Temperature: 20˜26° C.    -   Humidity 40-70%.        Cages: Made of polycarbonate. The size is 300 mm×180 mm×150 mm.        The bedding material is corn cob, which is changed twice per        week.        Diet: Animals had free access to irradiation sterilized dry        granule food during the entire study period.        Water: Animals had free access to sterile drinking water.        Cage identification: The identification labels for each cage        contained the following information: number of animals, sex,        strain, the date received, treatment, study number, group number        and the starting date of the treatment.        Animal identification: Animals were marked by ear coding.        4. Experimental Methods and Procedures        4.1 Cell Culture

The tumor cells were maintained in Leibovitz's L-15 medium supplementedwith 10% heat inactivated fetal bovine serum at 37° C. in an atmosphereof 0% CO₂ in air. The tumor cells were routinely subcultured twiceweekly. The cells growing in an exponential growth phase were harvestedand counted for tumor inoculation.

4.2. Tumor Inoculation

Each mouse was inoculated subcutaneously at the right flank withMDA-MB-468 tumor cells (10×10⁶) in 0.2 ml of PBS supplemented with 50%matrigel for tumor development. 20 animals were randomized when theaverage tumor volume reached 464 mm³. The test article administrationand the animal numbers in each group were shown in the experimentaldesign table.

4.3. Testing Article Formulation Preparation

Test Conc. article Purity (mg/ml) Formulation Vehicle — — 25 mMHistidine pH 7 10% sucrose BCY8245 99.7% 1 Dissolve 5.0 mg BCY8245 in4.985 ml Histidine buffer¹ 0.1 Dilute 140 μl 1 mg/ml BCY8245 stock with1260 μl Histidine buffer 0.3 Dilute 420 μl 1 mg/ml BCY8245 stock with980 μl Histidine buffer BCY8234 98.10% 30 Dissolve 147 mg BCY8234 in4.807 ml Histidine buffer ¹25 mM Histidine pH 7 10% sucrose4.4. Sample Collection

At the end of the study, the tumors of group 3 was collected for FFPE.

5. Results

5.1. Tumor Growth Curve

The tumor growth curve is shown in FIG. 25.

5.2. Tumor Volume Trace

Mean tumor volume over time in female Balb/c nude mice bearingMDA-MB-468 xenograft is shown in Tables 40 to 42.

5.3. Tumor Growth Inhibition Analysis

Tumor growth inhibition rate for test articles in the MDA-MB-468xenograft model was calculated based on tumor volume measurements at day28 after the start of the treatment.

6. Results Summary and Discussion

In this study, the therapeutic efficacy of test articles in theMDA-MB-468 xenograft model was evaluated. The measured tumor volumes ofall treatment groups at various time points is shown in FIG. 25 andTables 40 to 43.

The initial tumor starting size was intentionally greater than thatpreviously used to determine whether BCY8245 showed efficacy in thislarger size. The mean tumor size of vehicle treated mice reached 773 mm³on day 28. BCY8245 at 1 mg/kg, qw (TV=384 mm³, TGI=126.6%, p<0.001) and3 mg/kg, qw (TV=50 mm³, TGI=234.6%, p<0.001) produced significantanti-tumor activity in dose dependent manner on day 28. Among them, themice treated with BCY8245, 3 mg/kg qw showed some tumor relapse afterceasing the treatment, the further dosing from day 76 didn't work oncomplete tumor regression.

BCY8245 at 3 mg/kg, qw in combination with BCY8234 300 mg/kg, qwproduced significant anti-tumor activity (TV=55 mm³, TGI=234.0%,p<0.001) on day 28, and the tumors didn't showed any relapse during thewhole monitoring schedule.

The mice of vehicle group treated with 10 mg/kg Nectin-4 ADC or 5 mg/kgBCY8245 and the mice of group 2 (BCY8245, 1 mpk, qw) treated with 5mg/kg BCY8245 on PG-D28 showed effective tumor regression in thefollowing 3 weeks, after then, the tumors showed regrowth in the next 4weeks when taken off drug.

BCY8245 was able to cause tumor regression in the tumors ofapproximately 450 mm³, but also when administered to the grouppreviously receiving vehicle, in tumours with a starting volume ofapproximately 770 mm³.

TABLE 40 Tumor volume trace over time (Day 0 to day 28) Days after thestart of treatment Gr. Treatment 0 2 5 7 9 12 1 Vehicle, qw 466 ± 89 494± 94  529 ± 106  548 ± 109  574 ± 117  602 ± 130 2 BCY8245 466 ± 22 480± 24 453 ± 29 474 ± 25 446 ± 31 461 ± 34 1 mpk, qw 3 BCY8245 464 ± 28451 ± 24 388 ± 25 333 ± 30 284 ± 26 168 ± 24 3 mpk, qw 4 BCY8245 + 467 ±45 457 ± 46 401 ± 47 389 ± 42 309 ± 25 205 ± 9  BCY8234 (3 + 300) mpk,qw Days after the start of treatment Gr. 14 16 19 21 23 26 28 1  632 ±133  659 ± 129  686 ± 133  706 ± 145  741 ± 148  769 ± 157  773 ± 155 2460 ± 28 433 ± 37 412 ± 32 430 ± 32 421 ± 34 382 ± 37 384 ± 41 3 129 ±20  93 ± 23  83 ± 17  71 ± 19  60 ± 17  49 ± 17  50 ± 17 4 150 ± 9  125± 5  95 ± 3 90 ± 5 78 ± 5 66 ± 4 55 ± 5

TABLE 41 Tumor volume trace over time (Day 30 to day 75) Group1 Group 2,Group 4, Days Vehicle, BCY8245, BCY8245 + after the dosed with 1 mpk,change Group3, BCY8234 start of 5 mpk BCY8245 in 5 mpk BCY8245, 3 + 300treatment on PG-D28 on PG-D28 3 mpk, qw mpk, qw 30 625 ± 27  351 ± 42 44± 18 49 ± 8  33 477 ± 31  213 ± 29 43 ± 20 33 ± 10 35 405 ± 65  151 ± 1844 ± 20 31 ± 10 37 237 ± 36   98 ± 16 50 ± 24 31 ± 10 40 148 ± 33   89 ±17 55 ± 29 36 ± 14 42 142 ± 33   95 ± 16 66 ± 32 40 ± 13 44 132 ± 69 103 ± 20 71 ± 33 35 ± 11 48 146 ± 100 106 ± 21 80 ± 36 43 ± 14 51 171 ±122 103 ± 21 91 ± 43 45 ± 18 55 227 ± 166 104 ± 20 108 ± 53  42 ± 13 56264 ± 182 120 ± 23 125 ± 58  43 ± 12 62 288 ± 206 145 ± 29 146 ± 70  40± 12 65 316 ± 212 163 ± 31 147 ± 74  41 ± 14 68 347 ± 215 173 ± 32 155 ±81  46 ± 13 72 368 ± 242 180 ± 36 170 ± 89  45 ± 20 75 385 ± 245 196 ±40 223 ± 115 43 ± 19

TABLE 42 Tumor volume trace over time (Day 79 to day 103) Days after thestart of treatment Gr. Treatment 79 82 86 89 93 96 100 103 3 BCY8245 221± 118 198 ± 107 185 ± 105 180 ± 102 155 ± 91 166 ± 95 221 ± 119 250 ±125 3 mpk, qw

TABLE 43 Tumor growth inhibition analysis Tumor P value Volume T/C^(b)TGI compared Gr Treatment (mm³)^(a) (%) (%) with vehicle 1 Vehicle, qw773 ± 155 — — — 2 BCY8245 384 ± 41  49.7 126.6 p < 0.001 1 mpk, qw 3BCY8245 50 ± 17 6.4 234.6 p < 0.001 3 mpk, qw 4 BCY8245 + 55 ± 5  7.1234.0 p < 0.001 BCY8234 3 + 300 mpk, qw ^(a)Mean ± SEM. ^(b)Tumor GrowthInhibition is calculated by dividing the group average tumor volume forthe treated group by the group average tumor volume for the controlgroup (T/C).

Example 10: In Vivo PK Studies

MDA-MB-468 xenograft animals were injected with BCY8245 (BT8009) at 3mg/kg. At various timepoints, animals were euthanized and plasma andtumour taken and snap frozen. Samples were analysed for MMAE. The plasmalevels of BT8009 (BCY8245) are from historical PK studies. Theconcentrations of MMAE in plasma, MMAE in tumor, and BT8009 in plasmaare shown in FIG. 33. MMAE was retained in the tumour longer than inplasma supporting the hypothesis that systemic exposure is significantlyless than tumour exposure.

Example 11: HCS Assay

HCS assay was used in Nectin-4 BDC binding study. Cells were incubatedwith test agent and then washed. Detection was by a fluorescent antibodyto MMAE. MDA-MB-468 cells show moderate Nectin-4 expression with 20000cells giving the best images. NCI-H292 cells show low expression in thisassay, detection of MMAE was poor even at 20000 cells. HCS Data onMDA-MB-468 cell line is shown in FIG. 34, and Table 44.

TABLE 44 Max fluorescent Kd Historical Kd Test Item intensity (nM) (nM)Nectin-4 ADC 33.63 0.2 0.28 ± 0.07 BCY8245 13.34 3.52 5.18 MMAE2.95 >10000 >10000

Nectin-4 ADC and BCY8245 were retained on the cells and co-localisedwith a membrane stain. BCY8781 and MMAE showed minimal retention. Kd ofall compounds on MDA-MB-468 cell line were consistent with historicaldata. The Nectin-4 ADC showed detectable binding affinity on MDA-MB-468cell line. BCY8425 showed single digit nanomolar affinity with a Bmaxlower than for the Nectin-4 ADC. This reduced maximum fluorescentintensity is because the Nectin-4 ADC has an MMAE to drug ration of 4whereas BCY8245 has an MMAE to drug ratio of 1. BCY8781 showed only veryweak binding affinity on MDA-MB-468 cell line whilst MMAE showed almostno detectable binding affinity on MDA-MB-468 cell line.

Example 12: In Vivo Efficacy of BCY8245 in Two PDX Models of Lung Cancer

Purpose

To evaluate the efficacy of BCY8245 in a PDX model of squamous cellnon-small and adenocarcinoma (both non-small cell carcinomas).

Animals

-   -   Species: Mus Musculus    -   Strain: Balb/c nude    -   Age: 6-8 weeks    -   Sex: female    -   Body weight: 18-22        Agents in Test

BCY8245 and Nectin-4 ADC or BCY8781

Pre-Study Animals

Each mouse was inoculated subcutaneously at the right flank withLU-01-0007 or LU-01-0412 tumor fragment (˜30 mm³) for tumor development.Animals were randomized when the average tumor volume reached 161 mm³(LU-01-0007) or 147 mm³ (LU-01-0412)

In life Measurements and the Endpoints

Animals were checked daily for any effects of tumor growth andtreatments on normal behavior such as mobility, food and waterconsumption (by looking only), body weight gain/loss, eye/hair mattingand any other abnormal effect as stated in the protocol. Death andobserved clinical signs were recorded on the basis of the numbers ofanimals within each subset.

The major endpoint was to see if the tumor growth could be delayed ormice could be cured. Tumor volume was measured three times weekly in twodimensions using a caliper, and the volume was expressed in mm³ usingthe formula: V=0.5 a×b² where a and b are the long and short diametersof the tumor, respectively. The tumor size was then used forcalculations of T/C value. The T/C value (in percent) is an indicationof antitumor effectiveness; T and C are the mean volumes of the treatedand control groups, respectively, on a given day.

TGI was calculated for each group using the formula: TGI(%)=[1−(T_(i)−T₀)/(V_(i)−V₀)]×100; T_(i) is the average tumor volume ofa treatment group on a given day, T₀ is the average tumor volume of thetreatment group on the day of treatment start, Vi is the average tumorvolume of the vehicle control group on the same day with T_(i), and V₀is the average tumor volume of the vehicle group on the day of treatmentstart.

The results of these studies are shown in FIGS. 26 and 27.

Lu-01-0412 (FIG. 26): BCY8245 produced a dose related efficacy in thisPDX model with a reduction in tumour growth rate at 1 mg/kg qw butmarked tumor regression at 3 mg/kg qw to baseline. After cessation ofdosing (Day 21) 5/6 animals showed no tumour regrowth out to 105 dayspost study start. The single animal showing regrowth was responsive to 3mg/kg BCY8245 and showed restored regression to baseline. BCY8781 thenon-binding BDC produced stable disease at 3 mg/kg and on cessation ofdosing tumor rapidly grew at the same rate as the vehicle treated group,emphasising the increased efficacy that Nectin-4 binding affords theseagents. Large tumors (the vehicle treated group) regressed in responseto a single dose of BCY8245 or BCY8781.

LU-01-0007 (FIG. 27): BCY8245 produced a dose related efficacy with 1mg/kg qw producing stable disease and 3 mg/kg producing full regression.Dosing had to be maintained out to day 56 to attain full regression(when dosing was ceased). There was no tumor regrowth in this group outto beyond (the latter being maintained out to 126 days post studystart). The Nectin-4 ADC gave a similar degree of efficacy. The 1 mg/kgstable disease group was responsive to increases in dosing (3 and 5mg/kg) suggesting that low doses of BCY8245 do not lead to a developmentof resistance.

Example 13: In Vivo Evaluation of BCY8245 in Low Passage PDX Models ofHuman Breast, Esophageal and Bladder Cancer in Immunocompromised Mice

Purpose

To evaluate the antitumor activity of Bicycle Agent in Low PassageChampions TumorGraft Models of Human Breast, Esophageal, and BladderCancer in Immunocompromised mice.

Test System

Species: Mouse

Strain: Athymic Nude-Foxn1nu (Immune-compromised)

Source: Envigo: Indianapolis, Ind.

Gender: Female

Target age at initiation of dosing: At least 6-8 weeks of age

Target weight at initiation of dosing: At least 18 grams

Acclimation period: 3 days

Experimental Design

Pre-study Animals: When sufficient stock animals reach 1.0-1.5 cm³,tumors will be harvested for re-implantation into pre-study animals.Pre-study animals will be implanted unilaterally on the left flank withtumor fragments harvested from stock animals. Each animal is implantedfrom a specific passage lot and documented.

Study Animals: Pre-study tumor volumes are recorded for each experimentbeginning seven to ten days after implantation. When tumors reach anaverage tumor volume of 150-300 mm³, animals will be matched by tumorvolume into treatment or control groups to be used for dosing and dosinginitiated on Day 0.

Agents in Test

BCY8245 and Nectin-4 Antibody Drug conjugate, comparison with vehiclecontrol. Standard of care agent Docetaxel may be included. All agents tobe dosed qw by intravenous route, doses are indicated on graphs.

In Life Measurements

Efficacy Tumor Volume: Tumor volumes will be taken twice weekly. A finaltumor volume will be taken on the day study reaches endpoint. Ifpossible, a final tumor volume will be taken if an animal is foundmoribund.

Efficacy Animal Weights: Animals will be weighed twice weekly. A finalweight will be taken on the day the study reaches end point or if animalis found moribund, if possible. Animals exhibiting ≥10% weight loss whencompared to Day 0 will be provided DietGel® ad libitum. Any animalexhibiting >20% net weight loss for a period lasting 7 days or if micedisplay >30% net weight loss when compared to Day 0 will be consideredmoribund and euthanized.

Data Analysis

Agent Toxicity: Beginning on Day 0, animals will be observed daily andweighed twice weekly using a digital scale; data including individualand mean gram weights (Mean We±SEM), mean percent weight change versusDay 0 (% vD0) will be recorded for each group and % vD0 plotted at studycompletion. Animal deaths will be recorded daily and designated asdrug-related (D), technical (T), tumor related (B), or unknown (U) basedon weight loss and gross observation; single agent or combination groupsreporting a mean % vD0 >20% and/or >10% mortality will be consideredabove the maximum tolerated dose (MTD) for that treatment on theevaluated regimen. Maximum mean % vD0 (weight nadir) for each treatmentgroup is reported at study completion.

Agent Efficacy

Tumor Growth Inhibition—Beginning on Day 0, tumor dimensions aremeasured twice weekly by digital caliper and data including individualand mean estimated tumor volumes (Mean TV±SEM) recorded for each group;tumor volume is calculated using the formula (1): TV=width2×length×0.52.At study completion, percent tumor growth inhibition (% TGI) values willbe calculated and reported for each treatment group (T) versus control(C) using initial (i) and final (f) tumor measurements by the formula(2): % TGI=1−(Tf−Ti)/(Cf−Ci). Individual mice reporting a tumor volume≤30% of the Day 0 measurement for two consecutive measurements will beconsidered partial responders (PR). Individual mice lacking palpabletumors (0.00 mm³ for two consecutive measurements) will be classified ascomplete responders (CR); a CR that persists until study completion willbe considered a tumor-free survivor (TFS). Tumor doubling time (DT) willbe determined for the vehicle treated groups using the formulaDT=(Df−Di)*log 2/(log TVf−log TVi) where D=Day and TV=Tumor Volume. Alldata collected in this study is managed electronically and stored on aredundant server system.

The results of these studies are shown in FIGS. 28 to 31.

BCY8245 was tested in four low passage PDX models representing bladdercancer (CTG-1771), an estrogen and progesterone negative Her2 positivebreast cancer (CTG-1171) a triple negative breast cancer (CTG-1106) andan esophageal cancer (CTG-0896). In all of these models BCY8245 showedexcellent efficacy evoking tumor regression and in three of the fourfull regression to baseline. Efficacy was comparable to the ADC in allcases and superior or equal to Docetaxel SOC. In all models BCY8245 wasbetter tolerated than Docetaxel.

The invention claimed is:
 1. A peptide ligand specific for Nectin-4comprising a polypeptide comprising at least three cysteine residues,separated by at least two loop sequences, and a molecular scaffold whichforms covalent bonds with the cysteine residues of the polypeptide suchthat at least two polypeptide loops are formed on the molecularscaffold, wherein the peptide ligand comprises the amino acid sequence:(SEQ ID NO: 1) C_(i)P[1Nal][dD]C_(ii)M[HArg]DWSTP[HyP]WC_(iii);

wherein 1Nal represents 1-naphthylalanine, HArg represents homoarginine,HyP represents hydroxyproline and C_(i), C_(ii) and C_(iii) representfirst, second and third cysteine residues, respectively, or apharmaceutically acceptable salt thereof.
 2. The peptide ligandaccording to claim 1, which comprises an amino acid sequence selectedfrom: [B-Ala][Sar10]-(SEQ ID NO: 1) (hereinafterreferred to as BCY8234); Ac[B-Ala][Sar₅]-(SEQ ID NO: 1) (hereinafterreferred to as BCY8122); Ac-(SEQ ID NO: 1) (hereinafter referred to asBCY8126); (SEQ ID NO: 1) (hereinafter referred to as BCY8116);Fluorescein-(SEQ ID NO: 1) (hereinafter referred to as BCY8205); and[PYA][B-Ala][Sar10]-(SEQ ID NO: 1) (hereinafter referred to as BCY8846).


3. The peptide ligand according to claim 1, which comprises an aminoacid sequence selected from: [B-Ala][Sarl0]-(SEQ ID NO: 1) (hereinafterreferred to as BCY8234); Ac-[B-Ala][Sar₅]-(SEQ ID NO: 1) (hereinafterreferred to as BCY8122); Ac-(SEQ ID NO: 1) (hereinafter referred to asBCY8126); (SEQ ID NO: 1) (hereinafter referred to as BCY8116); andFluorescein-(SEQ ID NO: 1) (hereinafter referred to as BCY8205).


4. The peptide ligand according to claim 1, which comprises an aminoacid sequence selected from:Ac-[B-Ala][Sar₅]-(SEQ ID NO: 1) (hereinafter referred to as BCY8122);Ac-(SEQ ID NO: 1) (hereinafter referred to as BCY8126); and(SEQ ID NO: 1) (hereinafter referred to as BCY8116).


5. The peptide ligand according to claim 1, which comprises an aminoacid sequence selected from:Ac-[B-Ala][Sar₅]-(SEQ ID NO: 1) (hereinafter referred to as BCY8122);and Ac-(SEQ ID NO: 1) (hereinafter referred to as BCY8126).


6. The peptide ligand according to claim 1, which comprises an aminoacid sequence: [B-Ala][Sar10]-(SEQ ID NO: 1) (hereinafterreferred to as BCY8234).


7. The peptide ligand according to claim 1, wherein the molecularscaffold is 1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one(TATA).
 8. The peptide ligand according to claim 1, wherein thepharmaceutically acceptable salt is selected from the free acid or thesodium, potassium, calcium, or ammonium salt.
 9. The peptide ligandaccording to claim 1, wherein the Nectin-4 is human Nectin-4.
 10. Apharmaceutical composition which comprises the peptide ligand of claim1, in combination with one or more pharmaceutically acceptableexcipients.
 11. The pharmaceutical composition as defined in claim 10,which additionally comprises one or more therapeutic agents.
 12. A drugconjugate comprising a peptide ligand as defined in claim 1, conjugatedto one or more effector and/or functional groups.
 13. The drug conjugateas defined in claim 12, conjugated to one or more cytotoxic agents. 14.The drug conjugate as defined in claim 13, wherein said cytotoxic agentis selected from Monomethyl auristatin E (MMAE) and Mertansine (DM1).15. The drug conjugate as defined in claim 14, wherein the cytotoxicagent is MMAE and said conjugate additionally comprises a linkerselected from: -PABC-Cit-Val-Glutaryl- and-PABC-cyclobutyl-Ala-Cit-βAla-, wherein PABC representsp-aminobenzylcarbamate.
 16. The drug conjugate as defined in claim 15,wherein the cytotoxic agent is DM1 and said conjugate additionallycomprises a linker which is -SPDB-(SO₃H)—, wherein SPDB representsN-succinimidyl 3-(2-pyridyldithio)propionate.
 17. The drug conjugate asdefined in claim 15, wherein the linker is -PABC-Cit-Val-Glutaryl-. 18.The drug conjugate as defined in claim 12, which is selected from:BCY8245 and BCY8549.
 19. The drug conjugate as defined in claim 18,which is BCY8245.
 20. The drug conjugate as defined in claim 14, whereinsaid cytotoxic agent is MMAE.
 21. A pharmaceutical composition whichcomprises the drug conjugate of claim 12, in combination with one ormore pharmaceutically acceptable excipients.
 22. A method forsuppressing or treating a disease or disorder mediated by Nectin-4,comprising administering to a patient in need thereof the drug conjugateas defined in claim
 12. 23. The method of claim 22, wherein the diseaseor disorder mediated by Nectin-4 is selected from bladder cancer, breastcancer, colorectal cancer, lung cancer, ovarian cancer, and pancreaticcancer.
 24. A method of suppressing or treating a cancer patient havingan increased copy number variation (CNV) of Nectin-4, which comprisesadministering to said patient in need thereof the drug conjugate asdefined in claim 12.